CN113395908A - 包含巨大芽孢杆菌属益生菌菌株和多不饱和脂肪酸组分的制剂 - Google Patents

包含巨大芽孢杆菌属益生菌菌株和多不饱和脂肪酸组分的制剂 Download PDF

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CN113395908A
CN113395908A CN201980078589.8A CN201980078589A CN113395908A CN 113395908 A CN113395908 A CN 113395908A CN 201980078589 A CN201980078589 A CN 201980078589A CN 113395908 A CN113395908 A CN 113395908A
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omega
acid
fatty acid
bacillus megaterium
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B·施佩克曼
I·奥克龙贝尔
M·席林
M·戈梅兹
M·施旺
S·佩尔策
J·克莱因波特
T·柏格鲁伯
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Evonik Operations GmbH
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Abstract

本发明涉及一种制剂，其包含至少一种属于巨大芽孢杆菌属的益生菌菌株和多不饱和脂肪酸组分，所述多不饱和脂肪酸组分包含选自二十碳五烯酸(EPA)、二十二碳六烯酸(DHA)、花生四烯酸(ARA)、α亚麻酸、亚麻油酸、二十碳四烯酸、二十二碳五烯酸、亚油酸、γ‑亚麻酸的至少一种ω‑3或ω‑6脂肪酸，其中所述多不饱和脂肪酸组分包含ω‑3或ω‑6脂肪酸盐。本发明进一步涉及本发明的制剂作为饲料或食物补充剂、药物产品、饲料或食品组合物以及用于局部应用的用途。

Description

包含巨大芽孢杆菌属益生菌菌株和多不饱和脂肪酸组分的 制剂
本发明涉及一种制剂，其包含至少一种属于巨大芽孢杆菌(Bacillusmegaterium)属的益生菌菌株或巨大芽孢杆菌的提取物，以及多不饱和脂肪酸组分，所述多不饱和脂肪酸组分包含选自二十碳五烯酸(EPA)、二十二碳六烯酸(DHA)、花生四烯酸(ARA)、α亚麻酸、亚麻油酸(stearidonic acid)、二十碳四烯酸、二十二碳五烯酸、亚油酸、γ-亚麻酸和/或其衍生物的至少一种ω-3或ω-6脂肪酸。
ω-3脂肪酸，即α-亚油酸(ALA)、EPA和DHA的饮食摄入对人体健康特别是对例如类风湿关节炎的改善和心血管疾病危险因素的减少有益[1、2]。各种海鲜产品是饮食中EPA/DHA的来源，但其消耗量通常不足以满足建议的饮食限额(通常每天500mg EPA和DHA)[3]。这一缺口通过广泛使用含有ω-3脂肪酸的膳食补充剂或强化食物来弥合[4]。膳食补充剂是营养物质或具有营养或生理作用的其他物质的集中来源，其目的是补充正常饮食(www.efsa.europa.eu/en/topics/topic/food-supplements)。例如，ω-3脂肪酸补充剂通常含有来自鱼油、磷虾油或藻类的EPA/DHA的甘油三酯或ω-3乙酯。
ω-3脂肪酸通常具有抗炎作用、心脏和神经保护作用[2、5]。它们的作用方式包括直接清除活性氧，改变细胞膜流动性(其继而影响细胞信号转导事件)，调节转录因子诸如PPARG和NFKappaB(其安排促炎和抗炎细胞因子的生物合成)的活性，和竞争性排斥通过环氧合酶和脂氧合酶转化为促炎细胞因子的基质。
最近，已鉴定出ω-3和ω-6脂肪酸的几种氧合产物，并将其功能表征为其有益的健康作用的关键介质，特别是在改善慢性炎症病况方面[6]。这些产品包括maresin(MaR)、E-和D-系列消退素(RvE和RvD)、保护素、脂氧素及其前体，例如18-羟基-二十碳五烯酸(18-HEPE)、17-羟基-二十二碳六烯酸(17-HDHA)和17,18-环氧二十碳四烯酸(17,18-EEQ)，统称为特化促炎症消退(pro-resolving)脂质介质(SPM)。SPM由脂氧合酶、环氧合酶2和细胞色素P450单加氧酶(CYP450)内源性形成，并充当活性炎症消退的有效激动剂，通过纳摩尔浓度的G蛋白偶联受体进行信号传导。在使用啮齿动物的研究中证明了SPM针对多种传染性和炎性疾病的有效性[6]。例如，RvE1、RvD2、保护素D1(PD1)和LXA4增强致病性牙龈假单胞菌(Pseudomonas gingivalis)[7]、大肠杆菌(E.coli)[8]、单纯疱疹(Herpes simples)[9]、念珠菌(Candida)[10]、H5N1流感[11]的清除率。
LXA4、LXB4、RvE1、RvE3、RvD1-5、RvD2、PD1、MaR1、MaR2在牙周炎、囊性纤维化、神经炎症、缺血性中风、阿尔茨海默病[12]、动脉粥样硬化[13]、非酒精性脂肪肝疾病[14]、角膜损伤[15]、视网膜病变[16]、青光眼[17]、结肠炎[18]、哮喘[19、20]、胰岛素抗性[14]、关节炎[21]和疼痛[22]的模型中具有保护作用。此外，SPM的几种前体本身已显示发挥消退作用。例如，18-羟基-二十碳五烯酸(18-HEPE)通过抑制单核细胞与血管内皮细胞的粘附[23]和通过抑制压力超负荷诱发的适应不良的心脏重塑[24]来抵消心血管疾病的发展。类似地，17,18-EEQ具有心脏保护、抗心律不齐、血管舒张和抗炎特性[5]。肠神经胶质细胞的ARA衍生的15-HETE的旁分泌支持肠屏障功能，其是在例如克罗恩病中受到损害的过程[25]。
然而，将这些有希望的临床前发现结果转化用于改善人类健康已显示具有挑战性。如在实验研究中所做的那样通过静脉内或腹膜内注射直接递送SPM对人类而言是不可行的，特别是在预防方法的情况下。含SPM或SPM前体的补充剂或食物的口服递送是不合理的，因为它们在生物体液中的半衰期相对较短，因此它们不太可能到达其靶组织。在这方面，WO 2017/041094公开了一种浓缩的酯化鱼油，其仅包含约0.0005％的18-HEPE和17-HDHA，并且甚至通过超临界流体萃取富集这些SPM前体也不会产生超过0.05％(18-HEPE+17-HDHA)/总ω-3。
使用EPA/DHA的临床试验得出不确定的或无效的结果，特别是对于患有炎症性肠病、哮喘和代谢综合征特征的患者[2]。这种对人类的益处的缺乏与SPM对相应动物疾病模型的有效治疗形成了鲜明对比[6]。我们认为，将ω-3(和ω-6)转化为SPM是至关重要的步骤，这对于从旨在使用多不饱和脂肪酸(PUFA)预防、治疗或治疗炎性疾病的任何干预措施递送成功的结果至关重要。我们还认为，产生SPM的机构在某些条件下是功能失调的。这一想法得到了在糖尿病伤口[26]、代谢综合征[27]、哮喘[19、28]、溃疡性结肠炎[29]、克罗恩病[25]和牙周炎[30]中减少的(局部或循环)SPM水平以及严重哮喘[28]、溃疡性结肠炎[29]、囊性纤维化[31]、牙周炎[30]和阿尔茨海默氏病[12]中减少的SPM产生酶的表达或活性的发现结果的支持。
因此，本发明的目的是提供一种促进生物体内SPM形成的技术，从而在仅补充ω-3几乎没有或没有取得成功时，为遭受上述病况并且需要新的策略来预防、改善或治愈此类和类似病况的人和动物带来益处。
通过本发明，通过将合适的ω-3源与新发现的微生物SPM生产者在合适的制剂中组合来实现该目标，使得与单独的ω-3源相比，该组合技术提供增强的和/或靶向的功效。合适的制剂允许组分在小肠远端的胃肠道中伴随释放，从而防止ω-3源被小肠吸收，并使其更易于被微生物SPM生产者代谢。
SPM的生物合成已经针对真核细胞，特别是粒细胞和单核细胞进行了详细描述。巨噬细胞可以表达SPM生物合成所需的所有酶；其他仅表达选定酶的细胞类型可以与补体细胞一起表达。在肥大细胞中发现ALOX5，在皮肤和上皮细胞中发现ALOX12，在树突状细胞和肠神经胶质细胞中发现ALOX15[25]，在上皮细胞中发现COX-2和CYP450亚同种型。
鉴于肠道菌群决定个体对食物成分的反应并随后改变健康结果，我们将其鉴定为我们的促进内源性SPM生产的技术方法的靶标。靶向微生物群的策略通常包括应用益生元和益生菌，旨在改变微生物群的组成和活性。益生菌是活的微生物，当以足够的量施用时，其赋予宿主健康益处(FAO-WHO；Probiotics in food.Health and nutritionalproperties and guidelines for evaluation；FAO Food and Nutritional Paper 85,2006)。益生元支持有益微生物的生长。已经描述了ω-3脂肪酸的益生元作用[32、33]，但反之亦然，肠微生物对ω-3的可能的代谢影响尚待确定，并已在本发明中公开。肠道驻留微生物中耗氧酶的存在受到限制；胃肠道细菌和古细菌似乎不存在环氧合酶和脂氧合酶，但致病性铜绿假单胞菌(Pseudomonas aeruginosa)表达的15-脂氧合酶除外[34]。CYP450单加氧酶已在芽孢杆菌属中被检测到[35]。CYP102A1(也称为CYP450BM-3)是一种在巨大芽孢杆菌中发现的双功能酶，其通过连续的加氧酶和还原酶活性催化NADPH依赖性的多不饱和脂肪酸的羟基化反应。该P450系统由具有两个不同的结构域的多肽链组成，一个结构域包含血红蛋白，另一个结构域包含FAD还原酶。这种细菌细胞色素P450类别是可溶的，并通过[2Fe-2S]型铁硫蛋白从依赖于NADH的含FAD的还原酶获得反应机理所需的电子[36]。来源于表达载体构建体的纯化的CYP450BM-3已显示在无细胞反应中从EPA生成18-HEPE[37]。但是，这种反应在益生菌或合生元策略中的可能应用(其中野生型益生菌菌株是将细胞外EPA/DHA活化为18-HEPE或其他SPM的“催化剂”，并且更重要地使得这些分子可用于宿主)尚未被描述。此外，迄今为止，尚未描述任何其他益生菌微生物将ω-3或ω-6化合物氧化为任何其他SPM或生物活性脂质介质。
本发明涉及一种制剂，其包含至少一种属于巨大芽孢杆菌属的益生菌菌株和多不饱和脂肪酸组分，所述多不饱和脂肪酸组分包含选自二十碳五烯酸(EPA)、二十二碳六烯酸(DHA)、花生四烯酸(ARA)、α亚麻酸、亚麻油酸、二十碳四烯酸、二十二碳五烯酸、亚油酸、γ-亚麻酸的至少一种ω-3或ω-6脂肪酸，其中所述多不饱和脂肪酸组分包含ω-3或ω-6脂肪酸盐。
这种新的制剂促进了肠腔中各种SPM的形成，从而使它们可用于宿主并在其中发挥生理功能。从EPA/DHA生物合成SPM所需的氧气可从肠腔中获得：据报道，人类直肠中的气体含有0.3–1.8％的氧气[38]。此外，已经对小鼠描述了腔内氧气的径向分配，其朝着(血管化的)盲肠粘膜从1mmHg pO2增加到40mmHg pO2[39]，这表明与粘膜相关的耐氧微生物面临着相对富氧的环境，其允许氧依赖性生化反应。
发现巨大芽孢杆菌种的细菌特别适合于这种作用。因此，益生菌菌株包括该种的菌株。本发明的关键特征是菌株导致细胞外大量的SPM，这是引起对宿主的生理作用的先决条件。我们公开了纳摩尔水平上胞外SPM的巨大芽孢杆菌依赖性生产，借此一些SPM在皮摩尔水平上具有生理活性[6]，超过了对于人类血浆[27、40、41]和部分对于人类乳汁[42]的那些报道。根据本发明，使用整个细胞和裂解的细菌细胞(包括细菌细胞的所有组分)都是可行的。也可以使用细胞提取物。
最近在人类粪便[43]和唾液[44]样品中检测到巨大芽孢杆菌，表明这些细菌是人类肠道的居民。因此，本发明还涵盖了使用ω-3或ω-6组分来促进胃肠道微生物区系通过例如作为天然存在的肠道居住者的巨大芽孢杆菌种的菌株在胃肠道中形成各种SPM。
本发明菌株的细胞(特别是在本发明的组合物中)可以作为孢子(其是休眠的)、作为营养细胞(其正在生长)、作为过渡状态细胞(其从生长期过渡至孢子形成期)或作为至少两种特别是所有这些类型的细胞的组合存在。因此，在一个优选的实施方案中，益生菌菌株以休眠形式或作为营养细胞存在。
在一个优选的实施方案中，ω-3或ω-6脂肪酸是游离脂肪酸、盐、天然甘油三酯、鱼油、磷脂酯或乙酯的形式。
在另一个优选的配置中，所述脂肪酸选自ω-3脂肪酸EPA和DHA，或者其中ω-6脂肪酸组分为ARA。
本发明的另一种配置是任何上述组合物与5-氨基酮戊酸的组合，5-氨基酮戊酸是一种增强血红素生物合成的化合物[45]，并因此可以触发巨大芽孢杆菌的加氧酶活性。
在一个优选的实施方案中，益生菌菌株选自以下的一种或多种：巨大芽孢杆菌DSM32963、DSM 33296或DSM 33299。
通过筛选天然存在的分离株已鉴定出巨大芽孢杆菌DSM 32963、DSM 33296和DSM33299。它们已按照国际认可用于专利程序的微生物保藏的布达佩斯条约的规定，以EvonikDegussa GmbH的名义以上文提及的登录号于2018年11月27日(DSM 32963)和2019年10月17日保藏于DSMZ。
因此，用于根据本发明的制剂的巨大芽孢杆菌菌株选自以下组：
a)以DSM 32963、DSM 33296和DSM 33299保藏于DSMZ的巨大芽孢杆菌菌株之一；
b)以DSM 32963保藏的巨大芽孢杆菌菌株的突变体，其具有DSM 32963菌株的所有鉴定特征，其中所述突变体优选与DSM 32963菌株具有至少95％，优选至少96、97或98％，更优选至少99或99.5％的DNA序列同一性；
c)(a)或(b)的制剂；
d)包含如包含在(a)、(b)或(c)中的代谢物的有效混合物的制剂。
以DSM 32963保藏于DSMZ的巨大芽孢杆菌菌株表现出以下特征序列：
a)与根据SEQ ID NO：1或SEQ ID NO：2的多核苷酸序列具有至少99.5％(尤其是100％)的序列同一性的16S rDNA序列；
b)与根据SEQ ID NO：3的多核苷酸序列具有至少99.5％(尤其是100％)的序列同一性的yqfD序列；
c)与根据SEQ ID NO：4的多核苷酸序列具有至少99.5％(尤其是100％)的序列同一性的gyrB序列；
d)与根据SEQ ID NO：5的多核苷酸序列具有至少99.5％(尤其是100％)的序列同一性的rpoB序列；
e)与根据SEQ ID NO：6的多核苷酸序列具有至少99.5％(尤其是100％)的序列同一性的groEL序列。
以DSM 33296保藏于DSMZ的巨大芽孢杆菌菌株表现出以下特征序列：
a)与根据SEQ ID NO：13或SEQ ID NO：14的多核苷酸序列具有至少99.5％(尤其是100％)的序列同一性的16S rDNA序列；
b)与根据SEQ ID NO：15的多核苷酸序列具有至少99.5％(尤其是100％)的序列同一性的yqfD序列；
c)与根据SEQ ID NO：16的多核苷酸序列具有至少99.5％(尤其是100％)的序列同一性的gyrB序列；
d)与根据SEQ ID NO：17的多核苷酸序列具有至少99.5％(尤其是100％)的序列同一性的rpoB序列；
e)与根据SEQ ID NO：18的多核苷酸序列具有至少99.5％(尤其是100％)序列同一性的groEL序列。
以DSM 33299保藏于DSMZ的巨大芽孢杆菌菌株表现出以下特征序列：
a)与根据SEQ ID NO：25或SEQ ID NO：26的多核苷酸序列具有至少99.5％(尤其是100％)的序列同一性的16S rDNA序列；
b)与根据SEQ ID NO：27的多核苷酸序列具有至少99.5％(尤其是100％)的序列同一性的yqfD序列；
c)与根据SEQ ID NO：28的多核苷酸序列具有至少99.5％(尤其是100％)的序列同一性的gyrB序列；
d)与根据SEQ ID NO：29的多核苷酸序列具有至少99.5％、尤其是100％的序列同一性的rpoB序列；
e)与根据SEQ ID NO：30的多核苷酸序列具有至少99.5％(尤其是100％)的序列同一性的groEL序列。
因此，本发明的另一主题是巨大芽孢杆菌菌株，特别是如上文所述的巨大芽孢杆菌菌株，其表现出以下特征中的至少一个，优选全部：
a)与根据SEQ ID NO：1或SEQ ID NO：2、SEQ ID NO：13或SEQ ID NO：14、SEQ IDNO：25或SEQ ID NO：26的多核苷酸序列具有至少99％、优选至少99.5％、更优选至少99.8％或99.9％、尤其是100％的序列同一性的16S rDNA序列；
b)与根据SEQ ID NO：3、SEQ ID NO：15或SEQ ID NO：27的多核苷酸序列具有至少99％、优选至少99.5％、更优选至少99.8％或99.9％、尤其是100％的序列同一性的yqfD序列；
c)与根据SEQ ID NO：4、SEQ ID NO：16、SEQ ID NO：28的多核苷酸序列具有至少99％、优选至少99.5％、更优选至少99.8％或99.9％、尤其是100％的序列同一性的gyrB序列。
优选地，该巨大芽孢杆菌菌株表现出以下进一步的特征中的至少一个，更优选全部：
d)与根据SEQ ID NO：5、SEQ ID NO：17、SEQ ID NO：29的多核苷酸序列具有至少99％、优选至少99.5％、更优选至少99.8或99.9％、尤其是100％的序列同一性的rpoB序列；
e)与根据SEQ ID NO：6、SEQ ID NO：18、SEQ ID NO：30的多核苷酸序列具有至少99％、优选至少99.5％、更优选至少99.8％或99.9％、尤其是100％的序列同一性的groEL序列。
因此，本发明的一个特定主题还是巨大芽孢杆菌菌株，其表现出以下特征：
a)根据SEQ ID NO：1或SEQ ID NO：2、SEQ ID NO：13或SEQ ID NO：14或SEQ ID NO：25或SEQ ID NO：26的16S rDNA序列；
b)根据SEQ ID NO：3、SEQ ID NO：15或SEQ ID NO：27的yqfD序列；
c)根据SEQ ID NO：4、SEQ ID NO：16或SEQ ID NO：28的gyrB序列。
优选地，该巨大芽孢杆菌菌株表现出以下进一步的特征：
d)根据SEQ ID NO：5、SEQ ID NO：17或SEQ ID NO：29的rpoB序列；
e)根据SEQ ID NO：6、SEQ ID NO：18或SEQ ID NO：30的groEL序列。
在有利的配置中，EPA和DHA为游离脂肪酸、盐、天然甘油三酯、鱼油、磷脂酯或ω-3乙酯的形式。
EPA和DHA作为脂肪酸盐添加时，被益生菌菌株有效地转化为SPM，其生产和应用已在之前披露。WO2016102323A1描述了包含多不饱和ω-3脂肪酸盐的组合物，其可以抵抗氧化稳定存在。WO2017202935A1公开了一种制备包含ω-3脂肪酸盐和胺的组合物的方法，其中包含一种或多种ω-3脂肪酸、一种或多种碱性胺和20重量％或更少的水(基于糊剂的总重量)的糊剂被捏合直至获得均质的糊剂。
因此，在本发明的优选配置中，ω-3组分包含ω-3脂肪酸氨基酸盐，其中所述氨基酸选自碱性氨基酸，所述碱性氨基酸选自赖氨酸、精氨酸、鸟氨酸、组氨酸、瓜氨酸、胆碱及其混合物。
在另一个优选的配置中，氨基酸选择自选自赖氨酸、精氨酸、鸟氨酸、胆碱及其混合物的碱性氨基酸。
最优选使用赖氨酸的氨基酸盐。
本发明的另一优选配置是ω-3分散体(推测是脂质体)的制剂，以进一步提高对益生菌菌株的生物利用度。这样的分散体制剂优选由磷脂混合物(例如脱油的向日葵卵磷脂)或确定的磷脂例如二油基磷脂酰胆碱(DOPC)组成。这样的分散体制剂的最优选形式包含游离的ω-3脂肪酸盐或游离的ω-3脂肪酸。
因此，在该优选的实施方案中，多不饱和脂肪酸组分包括包含至少一种磷脂和至少一种ω-3脂肪酸的分散体的制剂。
在另一个优选的实施方案中，多不饱和脂肪酸组分包括包含至少一种磷脂和阳离子与衍生自ω-3或ω-6脂肪酸的阴离子的至少一种脂肪酸盐的分散体的制剂。特别优选使用ω-3脂肪酸。
在本发明的替代配置中，磷脂是脱油的磷脂，其包含大于40重量％、优选70重量％、更优选大于90重量％的磷脂酰胆碱含量和小于5重量％、优选小于1重量％的磷脂酰乙醇胺含量。
在一个替代的实施方案中，磷脂是油酸和/或亚油酸含量大于总脂肪酸的70重量％的非氢化磷脂。
在本发明的另一优选配置中，磷脂与脂肪酸盐的质量比大于0.001，优选大于0.05，更优选大于0.01，更优选大于0.09，最优选大于0.39。
在一个替代的实施方案中，所述制剂为粉末或液体形式，当与水在pH 6.5至7.5的pH值下混合时，其产生平均粒径小于1μm、优选小于500nm、最优选小于250nm的胶体分散体。
在另一个实施方案中，组分彼此精细分散，使得磷脂和脂肪酸盐均以100μg或更小的量存在和可检测到。
用于本发明制剂的肠内递送的优选制剂是提供针对胃病况的保护的制剂，或提供在小肠中制剂的靶向释放的制剂，或提供在大肠中制剂的靶向释放的制剂。因此，在优选的实施方案中，制剂包含用于延迟释放或肠或结肠释放的包衣。
本发明的一个主题是根据本发明的制剂作为饲料或食物补充剂的用途或其在食品中的用途。根据本发明的优选食品是巧克力产品、软糖、麦片、麦片条和乳制品。
本发明的另一个主题是本发明的制剂作为饲料或食物产品中的合益素成分的用途。
本发明的另一个主题是一种饲料或食品组合物，其包含根据本发明的制剂和至少一种另外的饲料或食物成分，优选地选自蛋白质、碳水化合物、脂肪、其他益生菌、益生元、酶、维生素、免疫调节剂、代乳品、矿物质、氨基酸、抗球虫药、基于酸的产品、药物及其组合。
根据本发明的饲料或食品组合物还包括丸剂、胶囊、片剂或液体形式的膳食补充剂。
本发明的另一个主题是药物组合物，其包含根据本发明的制剂和药学上可接受的载体。
本发明的另一主题是制剂在制备药物产品中的用途。
当将本发明的制剂施用于动物或人类时，优选改善健康状态，特别是动物或人类的肠道健康、心血管健康、心脏代谢健康、肺健康、关节健康、眼睛健康、精神健康、口腔健康或免疫健康。
因此，本发明的另一个主题是作为本发明的一部分的根据本发明的组合物，其用于改善健康状态，特别是动物或人类的肠道健康、心血管健康、心脏代谢健康、肺健康、关节健康、眼睛健康、精神健康、口腔健康或免疫健康。
根据本发明的有利配置是用于通过以下一种或多种改善动物或人的健康状态的组合物：
-通经由其通过胃肠道微生物的产生增加宿主中以下脂质介质的总量：
17-羟基-DHA(17-HDHA)，14-羟基-DHA(14-HDHA)，13-羟基-DHA(13-HDHA)，7-羟基-DHA(7-HDHA)，4-羟基-DHA(4-HDHA)，18-羟基-二十碳五烯酸(18-HEPE)，15-羟基-二十碳五烯酸(15-HEPE)，12-羟基-二十碳五烯酸(12-HEPE)，11-羟基-二十碳五烯酸(11-HEPE)，8-羟基-二十碳五烯酸(8-HEPE)，5-羟基-二十碳五烯酸(5-HEPE)，15-羟基-二十碳四烯酸(15-HETE)，12-羟基-二十碳四烯酸(12-HETE)，11-羟基-二十碳四烯酸(11-HETE)，8-羟基-二十碳四烯酸(8-HETE)，5-羟基-二十碳四烯酸(5-HETE)，9-羟基-十八碳二烯酸(9-HODE)，13-羟基-十八碳二烯酸酸(13-HODE)，19Z-二十二碳六烯酸(PDX)，保护素D1(PD1)，阿司匹林触发的PD1(AT-PD1)，马林素1(MaR1)，马林素2(MaR2)，白三烯B4(LTB4)，t-LTB4，消退素D1-5(RvD1-5)，阿司匹林触发的RvD1(AT-RvD1)，消退素E1(RvE1)，消退素E3(RvE3)，脂氧素A4(LXA4)，脂氧素A5(LXA5)，脂氧素B4(LXB4)，脂氧素B5(LXB5)，
-增加宿主中EPA的总量，
-增加宿主中DHA的总量，
本发明的另一个主题是本发明的制剂使用合适的基质或载体在对皮肤、眼睛和在口腔中的局部应用中的用途。
在优选的配置中，将制剂装载在包含纳米纤维素的预合成多相生物材料上和/或中，其中所述纳米纤维素是选自以下的细菌合成的纳米纤维素(BNC)：
-包含纤维素纤维或纳米晶须的网络的BNC，
-包含两个或更多个不同层的纤维素原纤维的BNC，其中每个层由来自不同微生物或来自在不同条件下培养的微生物的BNC组成，
-包含至少两个不同的纤维素网络的BNC，或
-BNC复合材料，其进一步包含聚合物。
纳米纤维素是指纳米结构纤维素的术语。它可以是纤维素纳米晶体(CNC或NCC)、也称为微纤化纤维素(MFC)的纤维素纳米纤维(CNF)或细菌纳米纤维素(BNC)，其是指由细菌产生的纳米结构纤维素。BNC是一种由诸如Komagataeibacter xylinus的菌株产生的纳米纤维聚合物，该Komagataeibacter xylinus是在纤维素生产中效率最高的最佳细菌种之一。BNC是具有独特的特性的生物材料，该特征为例如：化学纯度，出色的机械强度，高柔韧性，高吸收性，由于非凡的可成型性和柔软性而具有形成任何形状和尺寸的可能性等。此外，该材料是素食的和严格素食的，并且具有高水分含量。
细菌纤维素是三维网络，并且是固定和捕获微生物及其他物质的载体。固定的生物材料(包括微生物)用于生物活性代谢物(例如抗微生物剂、代谢生物活性物)的原位/体内生物合成，触发微生物和生物活性物的释放和/或用作用于发酵过程的固定微工厂。
本发明的另一主题还是表现出以下特征的休眠形式的或作为营养细胞的巨大芽孢杆菌菌株用于SPM的生物技术生产的用途：
a)与根据SEQ ID NO：1或SEQ ID NO：2、SEQ ID NO：13或SEQ ID NO：14、SEQ IDNO：25或SEQ ID NO：26的多核苷酸序列具有至少99.5％、尤其是100％的序列同一性的16SrDNA序列；和/或
b)与根据SEQ ID NO：3、SEQ ID NO：15或SEQ ID NO：27的多核苷酸序列具有至少99.5％、尤其是100％的序列同一性的yqfD序列；和/或
c)与根据SEQ ID NO：4、SEQ ID NO：16、SEQ ID NO：28的多核苷酸序列具有至少99.5％、尤其是100％的序列同一性的gyrB序列；和/或
d)与根据SEQ ID NO：5、SEQ ID NO：17、SEQ ID NO：29的多核苷酸序列具有至少99.5％、尤其是100％的序列同一性的rpoB序列；和/或
e)与根据SEQ ID NO：6、SEQ ID NO：18、SEQ ID NO：30的多核苷酸序列具有至少99.5％、尤其是100％的序列同一性的groEL序列。
工作实施例
实施例1：巨大芽孢杆菌菌株DSM 32963、DSM 33296和DSM 33299各自具有用于细 胞色素P450单加氧酶(CYP450)的遗传序列。
巨大芽孢杆菌菌株DSM 32963、DSM 33296和DSM 33299各自从来自威斯特伐利亚东部的原始花园的土壤样品中分离而来。它们已根据国际认可用于专利程序的微生物保存的布达佩斯条约的规定，以Evonik Degussa GmbH的名义以上文提及的登录号于2018年11月27日(DSM 32963)和2019年10月17日保藏于DSMZ。
巨大芽孢杆菌DSM 32963的基因组序列含有编码在氨基酸水平上与巨大芽孢杆菌ATCC 14581(AAA87602.1)的P450 BM3(CYP102A1)具有97.9％的同一性的蛋白质的基因[SEQ ID No：7]。该酶包含P450加氧酶和NADPH:P-450还原酶两者[46]。P450 BM3的天然底物经分析为长链脂肪酸(C12至C20)，其仅在近末端位置被羟基化(ω-1至ω-3)[47]。
与P450 BM3相比，对于在巨大芽孢杆菌DSM 32963的基因组序列内鉴定的序列观察到部分命中序列在氨基酸水平上21.1％至30.5％的较低序列相似性。与P450 BM3相比，这些其他潜在的细胞色素基因可能具有相似的功能[Seq ID No：8-12](参见表1)。
表1：针对巨大芽孢杆菌DSM 32963的蛋白质序列的P450 BM3的BLASTp(命中列中的SEQ ID是指相应的核苷酸序列)
Figure BDA0003089050030000141
巨大芽孢杆菌DSM 33296的基因组序列含有编码在氨基酸水平上与巨大芽孢杆菌ATCC 14581(AAA87602)的P450 BM3(CYP102A1)具有98.9％的同一性的蛋白质的基因[SEQID No：19]。该酶包含P450加氧酶和NADPH:P-450还原酶两者[46]。P450 BM3的天然底物经分析为长链脂肪酸(C12至C20)，其仅在近末端位置被羟基化(ω-1至ω-3)[47]。
与P450 BM3相比，对于在巨大芽孢杆菌DSM 33296的基因组序列内鉴定的序列观察到部分命中序列在氨基酸水平上21.5％至30.7％的较低序列相似性。与P450 BM3相比，这些其他潜在的细胞色素基因可能具有相似的功能[Seq ID No：20-24](参见表2)。
表2：针对巨大芽孢杆菌DSM 33296的蛋白质序列的P450 BM3的BLASTp(命中列中的SEQ ID是指相应的核苷酸序列)
Figure BDA0003089050030000151
巨大芽孢杆菌DSM 33299的基因组序列含有编码在氨基酸水平上与巨大芽孢杆菌ATCC 14581(AAA87602.1)的P450 BM3(CYP102A1)具有96.1％的同一性的蛋白质的基因[SEQ ID No：31]。该酶包含P450加氧酶和NADPH:P-450还原酶两者[46]。P450 BM3的天然底物经分析为长链脂肪酸(C12至C20)，其仅在近末端位置被羟基化(ω-1至ω-3)[47]。
与P450 BM3相比，对于在巨大芽孢杆菌DSM 33299的基因组序列内鉴定的序列观察到部分命中序列在氨基酸水平上20.9％至30.5％的较低序列相似性。与P450 BM3相比，这些其他潜在的细胞色素基因可能具有相似的功能[Seq ID No：32-37](参见表3)。
表3：针对巨大芽孢杆菌DSM 33299的蛋白质序列的P450 BM3的BLASTp(命中列中的SEQ ID是指相应的核苷酸序列)
Figure BDA0003089050030000161
实施例2：在磷酸盐缓冲液中用二油基磷脂酰胆碱(DOPC)制备ω-3脂肪酸分散体
为了制备ω-3脂肪酸分散体的制剂，将0.8g的二油基磷脂酰胆碱(DOPC，LipoidGmbH)溶解在1ml的乙醇中。添加并溶解0.2克鱼油(ω-3 1400，
Figure BDA0003089050030000162
)、ω-3乙酯(
Figure BDA0003089050030000163
500:200EE，BASF)或ω-3赖氨酸盐形式的游离ω-3脂肪酸的赖氨酸盐(
Figure BDA0003089050030000164
Evonik)。在游离的ω-3脂肪酸盐的情况下，添加20μl蒸馏水以完全溶解产品。
形式为ω-3赖氨酸盐的游离ω-3脂肪酸的赖氨酸盐(
Figure BDA0003089050030000165
Evonik)含有约67％的脂肪酸和大量的ω-3脂肪酸EPA和DHA，以及少量的ω-3脂肪酸二十二碳五烯酸和ω-6脂肪酸花生四烯酸、二十二碳四烯酸和二十二碳烯酸异构体。
在45℃的温度下和在剧烈搅拌下，将1ml的相应溶液逐滴加入到20ml的pH＝8的0.1M磷酸盐缓冲液中。之后，将分散体置于冰上并超声处理(Branson Sonifier，100％振幅，50％脉冲)15分钟，以产生纳米级分散体，推测是脂质体。最后，将分散体通过0.2μm注射器过滤器进行无菌过滤。通过动态光散射(DLS)测量(Zetasizer Nano ZS，Malvern)表征所得分散体的粒度。该分散体包含40g/l的磷脂和10g/l的ω-3脂肪酸或酯。
实施例3：巨大芽孢杆菌株菌DSM 32963能够产生细胞内18-羟基-二十碳五烯酸 (18-HEPE)
对于巨大芽孢杆菌DSM 32963，可以证明产生SPM的酶的相关的细胞内活性。从具有0.1％葡萄糖(LBG)的10ml Luria Bertami肉汤(LB，Thermo Fisher Scientific)中，将巨大芽孢杆菌DSM 32963的培养物在100ml培养瓶中于30℃和200rpm培养24h。将完整的培养物转移至LBG中的200ml主培养物中。主培养物在2升培养瓶中于30℃和200rpm生长6小时。然后以10ml的体积份收获细胞培养物，通过离心(15min，4000rpm，室温)除去上清液，并将细胞沉淀分别重悬于10ml LBG和2ml补充剂中(表2)。将这些培养物在100ml摇瓶中于30℃和200rpm孵育16小时。
将不同形式的二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)源添加到巨大芽孢杆菌DSM 32963细胞培养物中，以达到ω-3赖氨酸盐(Evonik的
Figure BDA0003089050030000171
)、鱼油(
Figure BDA0003089050030000172
的ω-3 1400)和ω-3乙酯(BASF的
Figure BDA0003089050030000173
500:200EE)的形式的0.4g/l的最终浓度。这些物质分别作为超声乳化液和分散体制剂(实施例2中所述的制剂)加入。
将不含ω-3脂肪酸含量的DOPC制剂和PBS缓冲液用作不含EPA的对照。
表2：补充剂、储液的制备及其EPA含量计算值(g/l)
Figure BDA0003089050030000181
通过离心(15分钟，4000rpm，室温)分离上清液，然后各自收获细胞培养物。然后，将上清液用由水/乙腈混合物组成的溶剂稀释(上清液:溶剂比例为1:2，溶剂组成：65％H2O，pH8和35％MeCN)。将沉淀冷冻干燥过夜，并重悬于由水/乙腈混合物组成的溶剂中(沉淀：溶剂比例为1:2，溶剂组成：65％H2O，pH8和35％MeCN)。细胞破碎是在Ribolyser中的Lysing Matrix管(0.1mm硅胶球)中进行的。
过滤细胞匀浆物(和稀释的上清液)，然后将其用于通过LC/ESI-MS分析(AgilentQQQ 6420，Gemini 3μC6-Phenyl)以阳性SIM模式在m/z 318下检测18-羟基-二十碳五烯酸(18-HEPE)，以及在m/z 302下检测前体化合物EPA。
将EPA以具有DOPC的ω-3赖氨酸盐、ω-3乙酯、ω-3赖氨酸盐分散体或具有DOPC的鱼油分散体的形式添加到巨大芽孢杆菌细胞中，导致18-HEPE的细胞相关(＝细胞内+细胞外吸附的)积累(表3)。迄今为止，通过添加ω-3赖氨酸盐可以实现细胞内18-HEPE的最高值；其是其他方法的十倍。
表3：巨大芽孢杆菌DSM 32963细胞的细胞提取物中细胞相关18-HEPE的浓度(mg/ml)
Figure BDA0003089050030000191
实施例4：巨大芽孢杆菌DSM 32963和ω-3脂肪酸盐
Figure BDA0003089050030000193
的分散体制剂的 合生元组合导致细胞外大量的18-HEPE
为了研究细胞外出现的18-HEPE的量，按照实施例1的描述培养巨大芽孢杆菌DSM32963细胞。将细胞重悬于10ml LBG或含9.76g/l FeSSIF-V2(biorelevant.com)(其是牛磺胆酸盐、磷脂和其他旨在模拟胆汁表面活性剂的组分的混合物)的LBG，并分别添加2ml的补充剂(表2)。另外，还将补充剂分别添加到没有细胞的摇瓶中的不同培养基中，并在相同条件下进行处理(对照)。在30℃和200rpm孵育16h后，测定培养上清液和对照的18-HEPE浓度(表4)。可以显示，巨大芽孢杆菌DSM 32963细胞能够从ω-3赖氨酸盐
Figure BDA0003089050030000192
分散体合成18-HEPE，其是可在细胞外检测的。值得注意的是，通过该方法检测的ω-3→18-HEPE的转化率高达0.075，这超过了WO 2017/041094公开的酯化鱼油中0.0005％的18-HEPE的基础含量。更重要的是，我们发现巨大芽孢杆菌菌株将ω-3赖氨酸盐转化为多种(最终)SPM产品，其浓度甚至高于18-HEPE(参见实施例6)，这是具有生理意义的。
表4：培养上清液和对照的测量的18-HEPE浓度(mg/l)
Figure BDA0003089050030000201
实施例5：不同芽孢杆菌属种对EPA的生物转化
为了研究不同芽孢杆菌属种产生细胞内18-HEPE的能力，按照实施例1所述培养不同种的细胞。将细胞重悬于10ml含9.76g/l FeSSIF-V2(biorelevant.com)(其是牛磺胆酸盐、磷脂和其他旨在模拟胆汁表面活性剂的组分的混合物)的LBG中，并分别添加1.2ml含DOPC的ω-3赖氨酸盐分散体。如实施例3中所述测定在30℃和200rpm孵育16小时后细胞的内部18-HEPE浓度。
仅巨大芽孢杆菌细胞能够从ω-3赖氨酸盐
Figure BDA0003089050030000202
分散体内部合成18-HEPE，而枯草芽孢杆菌(B.subtilis)、解淀粉芽孢杆菌(B.amyloliquefaciens)、短小芽孢杆菌(B.pumilus)和地衣芽孢杆菌(B.licheniformis)则不能。
表5：巨大芽孢杆菌DSM 32963细胞的细胞内测量的18-HEPE含量(mg/ml)
Figure BDA0003089050030000211
实施例6：在不同的培养条件下，巨大芽孢杆菌DSM 32963从ω-3脂肪酸盐
Figure BDA0003089050030000212
产生SPM
脂质代谢组学
细菌上清液样品经受使用RP相固相萃取的脂质提取，然后根据公开的程序[48]通过超高效液相色谱ESI串联质谱分析(UPLC-MS/MS)进行分析。在这些条件下，大约40种不同的LM(包括5-羟基-二十碳五烯酸5-HEPE，8-HEPE，11-HEPE，12-HEPE，15-HEPE，18-HEPE，5-羟基-二十碳四烯酸(5-HETE)，8-HETE，11-HETE，12-HETE，15-HETE，4-羟基-DHA(4-HDHA)，7-HDHA，13-HDHA，14-HDHA，17-HDHA，消退素A4(LXA4)，LXB4，消退素E1(RvE1)，RvE3，消退素D1-5(RvD1-5)，AT-RvD1，RvD2，protectin D1(PD1)，AT-PD1，maresin 1(MaR1)，MaR2)和4种脂肪酸底物可以以1pg的检测下限被检测到。
为了研究除了18-HEPE之外，巨大芽孢杆菌是否还能够产生其他PUFA氧合产物，进行来自如实施例4中所述培养的巨大芽孢杆菌菌株32963的具有
Figure BDA0003089050030000213
(溶解的或在分散体制剂中)的上清液(具有或不具有胆汁酸)的脂代谢组学筛选。并行处理和分析
Figure BDA0003089050030000214
制剂的无细胞制剂，并将其用作氧合产物的非酶促的自发形成的对照。表6中给出的值显示了形成的产物的净浓度，即减去对照值后。可以看出，通过细菌已经形成了包括SPM在内的许多氧合产物。几种SPM的浓度远远超过了人体血浆样品中发现的SPM浓度(其通常在约20-100pg/ml的范围内)[27、40、41]。为了进行比较，人母乳含有约6.000pg/mlRvE1和约10.000pg/ml RvD1[42]。考虑到SPM在nM或甚至pM范围内在啮齿动物中体内和体内地发挥受体介导的作用的事实[6]，表6中显示的发现结果强烈暗示了本发明的生理和治疗重要性。
PUFA制剂的类型对产物水平(其通常在存在PUFA分散体制剂和/或添加胆汁酸作为增溶剂的情况下更高)有很大的影响。并行地，与在没有分散体制剂和胆汁酸的情况下用AvailOm处理的样品相比，这些样品中单羟基化SPM前体5-HEPE、11-HEPE、12-HEPE、15-HEPE、18-HEPE、5-HETE、8-HETE和9-HODE的丰度较低。这可以通过这些前体向二羟基化和三羟基化脂肪酸的增加的转化来解释。
表6：巨大芽孢杆菌DSM 32963细胞的PUFA氧合产物的细胞外浓度
Figure BDA0003089050030000221
实施例7：通过其它巨大芽孢杆菌菌株从ω-3脂肪酸盐
Figure BDA0003089050030000222
的分散体制 剂产生SPM
筛选了来自不同生境的另外47种巨大芽孢杆菌菌株的SPM生产能力，以确定这是否是巨大芽孢杆菌种的普遍现象，并检测所生产的SPM的类型和数量的菌株特异性差异。如实施例4中所详述地培养细胞，其中
Figure BDA0003089050030000231
的分散体制剂用作ω-3脂肪酸源。并行地处理和分析
Figure BDA0003089050030000232
的无细胞制剂，并将其用作氧合产物的非酶促的自发形成的对照。观察到所有测试的菌株均产生可测量的(>1pg/ml)量的各种SPM及其前体，这些量在菌株之间差异极大(高达2.000倍)，并且RvE3的浓度在大多数菌株中特别高(高达1.3μg/ml)。
表7中给出的值显示了由两个表现最强的菌株(巨大芽孢杆菌DSM 33296和巨大芽孢杆菌DSM 33299)形成的PUFA氧合产物的净浓度。
表7：巨大芽孢杆菌DSM 33296和巨大芽孢杆菌DSM 33299细胞的PUFA氧合产物的细胞外浓度。
Figure BDA0003089050030000233
实施例8：包含EPA-DHA氨基酸盐和巨大芽孢杆菌菌株的胶囊作为食物补充剂或作 为药物
将以下组分填充入HPMC胶囊(00尺寸)中。
表7：用于填充到HPMC胶囊中的制剂。*氨基酸选自L-鸟氨酸、L-赖氨酸和L-精氨酸。#菌株选自巨大芽孢杆菌DSM 32963、DSM 33296、DSM 33299。
化合物 胶囊I 胶囊II 胶囊III
ω-3氨基酸*盐 250mg 50mg 800mg
巨大芽孢杆菌菌株<sup>#</sup> 1×10<sup>7</sup>CFU-1×10<sup>11</sup>CFU 1×10<sup>7</sup>CFU-1×10<sup>11</sup>CFU 1×10<sup>7</sup>CFU-1×10<sup>11</sup>CFU
胶囊可进一步包含选自L-鸟氨酸、L-天冬氨酸、L-赖氨酸和L-精氨酸的氨基酸。
胶囊可进一步包含选自阿拉伯木聚糖、大麦谷物纤维、燕麦谷物纤维、黑麦纤维、麦麸纤维、菊粉、低聚果糖(FOS)、低聚半乳糖(GOS)、抗性淀粉、β-葡聚糖、葡甘露聚糖、半乳葡甘露聚糖、瓜尔豆胶和低聚木糖的其他碳水化合物成分。
胶囊可进一步包含一种或多种植物提取物，其选自：姜、肉桂、葡萄柚、欧芹、姜黄、姜黄、橄榄果、人参、辣根、大蒜、西兰花、螺旋藻、石榴、花椰菜、羽衣甘蓝、香菜、绿茶、洋葱和水飞蓟。
胶囊可进一步包含虾青素、木炭、壳聚糖、谷胱甘肽、莫纳可林K、植物固醇、植物甾烷醇、萝卜硫烷、胶原蛋白、透明质酸、磷脂酰胆碱。
胶囊可包含选自生物素、维生素A、维生素B1(硫胺素)、维生素B2(核黄素)、维生素B3(烟酸)、维生素B5(泛酸)、维生素B9(叶酸或叶酸盐)、维生素C(抗坏血酸)、维生素D(钙化醇)、维生素E(生育酚和生育三烯酚)和维生素K(醌类)的其它维生素或选自硫、铁、氯、钙、铬、钴、铜、镁、锰、钼、碘、硒和锌的矿物质。
参考资料
1.Balk EM,Lichtenstein AH:Omega-3 Fatty Acids and CardiovascularDisease:Summary of the 2016Agency of Healthcare Research and Quality EvidenceReview.Nutrients 2017,9(8).
2.Calder PC:Marine omega-3 fatty acids and inflammatory processes:Effects,mechanisms and clinical relevance.Biochim Biophys Acta 2015,1851(4):469-484.
3.Papanikolaou Y,Brooks J,Reider C,Fulgoni VL,3rd:U.S.adults are notmeeting recommended levels for fish and omega-3 fatty acid intake:results ofan analysis using observational data from NHANES 2003-2008.Nutr J 2014,13:31.
4.Clarke TC,Black LI,Stussman BJ,Barnes PM,Nahin RL:Trends in the useof complementary health approaches among adults:United States,2002-2012.NatlHealth Stat Report 2015(79):1-16.
5.Schunck WH,Konkel A,Fischer R,Weylandt KH:Therapeutic potential ofomega-3 fatty acid-derived epoxyeicosanoids in cardiovascular andinflammatory diseases.Pharmacol Ther 2018,183:177-204.
6.Serhan CN,Krishnamoorthy S,Recchiuti A,Chiang N:Novel anti-inflammatory--pro-resolving mediators and their receptors.Curr Top Med Chem2011,11(6):629-647.
7.Serhan CN,Jain A,Marleau S,Clish C,Kantarci A,Behbehani B,ColganSP,Stahl GL,Merched A,Petasis NA et al:Reduced inflammation and tissue damagein transgenic rabbits overexpressing 15-lipoxygenase and endogenous anti-inflammatory lipid mediators.J Immunol 2003,171(12):6856-6865.
8.Prescott D,McKay DM:Aspirin-triggered lipoxin enhances macrophagephagocytosis of bacteria while inhibiting inflammatory cytokine production.AmJ Physiol Gastrointest Liver Physiol 2011,301(3):G487-497.
9.Rajasagi NK,Reddy PB,Mulik S,Gjorstrup P,Rouse BT:Neuroprotectin D1reduces the severity of herpes simplex virus-induced cornealimmunopathology.Invest Ophthalmol Vis Sci 2013,54(9):6269-6279.
10.Baillie JK,Digard P:Influenza--time to target the host？N Engl JMed 2013,369(2):191-193.
11.Morita M,Kuba K,Ichikawa A,Nakayama M,Katahira J,Iwamoto R,Watanebe T,Sakabe S,Daidoji T,Nakamura S et al:The lipid mediator protectinD1 inhibits influenza virus replication and improves severe influenza.Cell2013,153(1):112-125.
12.Zhu M,Wang X,Sun L,Schultzberg M,Hjorth E:Can inflammation beresolved in Alzheimer's disease？Ther Adv Neurol Disord 2018,11:1756286418791107.
13.Kasikara C,Doran AC,Cai B,Tabas I:The role of non-resolvinginflammation in atherosclerosis.J Clin Invest 2018,128(7):2713-2723.
14.Gonzalez-Periz A,Horrillo R,Ferre N,Gronert K,Dong B,Moran-Salvador E,Titos E,Martinez-Clemente M,Lopez-Parra M,Arroyo V et al:Obesity-induced insulin resistance and hepatic steatosis are alleviated by omega-3fatty acids:a role for resolvins and protectins.FASEB J 2009,23(6):1946-1957.
15.Jin Y,Arita M,Zhang Q,Saban DR,Chauhan SK,Chiang N,Serhan CN,DanaR:Anti-angiogenesis effect of the novel anti-inflammatory and pro-resolvinglipid mediators.Invest Ophthalmol Vis Sci 2009,50(10):4743-4752.
16.Connor KM,SanGiovanni JP,Lofqvist C,Aderman CM,Chen J,Higuchi A,Hong S,Pravda EA,Majchrzak S,Carper D et al:Increased dietary intake ofomega-3-polyunsaturated fatty acids reduces pathological retinalangiogenesis.Nat Med 2007,13(7):868-873.
17.Livne-Bar I,Wei J,Liu HH,Alqawlaq S,Won GJ,Tuccitto A,Gronert K,Flanagan JG,Sivak JM:Astrocyte-derived lipoxins A4 and B4 promoteneuroprotection from acute and chronic injury.J Clin Invest 2017,127(12):4403-4414.
18.Arita M,Yoshida M,Hong S,Tjonahen E,Glickman JN,Petasis NA,Blumberg RS,Serhan CN:Resolvin E1,an endogenous lipid mediator derived fromomega-3 eicosapentaenoic acid,protects against 2,4,6-trinitrobenzene sulfonicacid-induced colitis.Proc Natl Acad Sci U S A 2005,102(21):7671-7676.
19.Barnig C,Frossard N,Levy BD:Towards targeting resolution pathwaysof airway inflammation in asthma.Pharmacol Ther 2018,186:98-113.
20.Aoki H,Hisada T,Ishizuka T,Utsugi M,Kawata T,Shimizu Y,Okajima F,Dobashi K,Mori M:Resolvin E1 dampens airway inflammation andhyperresponsiveness in a murine model of asthma.Biochem Biophys Res Commun2008,367(2):509-515.
21.Perretti M,Norling LV:Actions of SPM in regulating host responsesin arthritis.Mol Aspects Med 2017,58:57-64.
22.Dalli J,Zhu M,Vlasenko NA,Deng B,Haeggstrom JZ,Petasis NA,SerhanCN:The novel 13S,14S-epoxy-maresin is converted by human macrophages tomaresin 1(MaR1),inhibits leukotriene A4 hydrolase(LTA4H),and shiftsmacrophage phenotype.FASEB J 2013,27(7):2573-2583.
23.Liu Y,Fang X,Zhang X,Huang J,He J,Peng L,Ye C,Wang Y,Xue F,Ai D etal:Metabolic profiling of murine plasma reveals eicosapentaenoic acidmetabolites protecting against endothelial activation and atherosclerosis.BrJ Pharmacol 2018,175(8):1190-1204.
24.Endo J,Sano M,Isobe Y,Fukuda K,Kang JX,Arai H,Arita M:18-HEPE,ann-3 fatty acid metabolite released by macrophages,prevents pressure overload-induced maladaptive cardiac remodeling.J Exp Med 2014,211(8):1673-1687.
25.Pochard C,Coquenlorge S,Jaulin J,Cenac N,Vergnolle N,Meurette G,Freyssinet M,Neunlist M,Rolli-Derkinderen M:Defects in 15-HETE Production andControl of Epithelial Permeability by Human Enteric Glial Cells From PatientsWith Crohn's Disease.Gastroenterology 2016,150(1):168-180.
26.Tang Y,Zhang MJ,Hellmann J,Kosuri M,Bhatnagar A,Spite M:Proresolution therapy for the treatment of delayed healing of diabeticwounds.Diabetes 2013,62(2):618-627.
27.Barden AE,Mas E,Croft KD,Phillips M,Mori TA:Specializedproresolving lipid mediators in humans with the metabolic syndrome after n-3fatty acids and aspirin.Am J Clin Nutr 2015,102(6):1357-1364.
28.Miyata J,Arita M:Role of omega-3 fatty acids and their metabolitesin asthma and allergic diseases.Allergol Int 2015,64(1):27-34.
29.Mangino MJ,Brounts L,Harms B,Heise C:Lipoxin biosynthesis ininflammatory bowel disease.Prostaglandins Other Lipid Mediat 2006,79(1-2):84-92.
30.Wang CW,Colas RA,Dalli J,Arnardottir HH,Nguyen D,Hasturk H,ChiangN,Van Dyke TE,Serhan CN:Maresin 1 Biosynthesis and Proresolving Anti-infective Functions with Human-Localized Aggressive PeriodontitisLeukocytes.Infect Immun 2015,84(3):658-665.
31.Ringholz FC,Buchanan PJ,Clarke DT,Millar RG,McDermott M,Linnane B,Harvey BJ,McNally P,Urbach V:Reduced 15-lipoxygenase 2 and lipoxin A4/leukotriene B4 ratio in children with cystic fibrosis.Eur Respir J 2014,44(2):394-404.
32.Robertson RC,Seira Oriach C,Murphy K,Moloney GM,Cryan JF,Dinan TG,Paul Ross R,Stanton C:Omega-3 polyunsaturated fatty acids critically regulatebehaviour and gut microbiota development in adolescence and adulthood.BrainBehav Immun 2017,59:21-37.
33.Mujico JR,Baccan GC,Gheorghe A,Diaz LE,Marcos A:Changes in gutmicrobiota due to supplemented fatty acids in diet-induced obese mice.Br JNutr 2013,110(4):711-720.
34.Vance RE,Hong S,Gronert K,Serhan CN,Mekalanos JJ:The opportunisticpathogen Pseudomonas aeruginosa carries a secretable arachidonate 15-lipoxygenase.Proc Natl Acad Sci U S A 2004,101(7):2135-2139.
35.Furuya T,Shibata D,Kino K:Phylogenetic analysis of Bacillus P450monooxygenases and evaluation of their activity towards steroids.Steroids2009,74(12):906-912.
36.Bernhardt R:Cytochromes P450 as versatile biocatalysts.JBiotechnol 2006,124(1):128-145.
37.Capdevila JH,Wei S,Helvig C,Falck JR,Belosludtsev Y,Truan G,Graham-Lorence SE,Peterson JA:The highly stereoselective oxidation ofpolyunsaturated fatty acids by cytochrome P450BM-3.J Biol Chem 1996,271(37):22663-22671.
38.Levitt MD:Volume and composition of human intestinal gasdetermined by means of an intestinal washout technic.N Engl J Med 1971,284(25):1394-1398.
39.Albenberg L,Esipova TV,Judge CP,Bittinger K,Chen J,Laughlin A,Grunberg S,Baldassano RN,Lewis JD,Li H et al:Correlation between intraluminaloxygen gradient and radial partitioning of intestinalmicrobiota.Gastroenterology 2014,147(5):1055-1063 e1058.
40.Polus A,Zapala B,Razny U,Gielicz A,Kiec-Wilk B,Malczewska-Malec M,Sanak M,Childs CE,Calder PC,Dembinska-Kiec A:Omega-3 fatty acidsupplementation influences the whole blood transcriptome in women withobesity,associated with pro-resolving lipid mediator production.BiochimBiophys Acta 2016,1861(11):1746-1755.
41.See VHL,Mas E,Prescott SL,Beilin LJ,Burrows S,Barden AE,Huang RC,Mori TA:Effects of prenatal n-3 fatty acid supplementation on offspringresolvins at birth and 12 years of age:a double-blind,randomised controlledclinical trial.Br J Nutr 2017,118(11):971-980.
42.Weiss GA,Troxler H,Klinke G,Rogler D,Braegger C,Hersberger M:Highlevels of anti-inflammatory and pro-resolving lipid mediators lipoxins andresolvins and declining docosahexaenoic acid levels in human milk during thefirst month of lactation.Lipids Health Dis 2013,12:89.
43.Piewngam P,Zheng Y,Nguyen TH,Dickey SW,Joo HS,Villaruz AE,GloseKA,Fisher EL,Hunt RL,Li B et al:Pathogen elimination by probiotic Bacillusvia signalling interference.Nature 2018,562(7728):532-537.
44.Al-Thubiani ASA,Maher YA,Fathi A,Abourehab MAS,Alarjah M,Khan MSA,Al-Ghamdi SB:Identification and characterization of a novel antimicrobialpeptide compound produced by Bacillus megaterium strain isolated from oralmicroflora.Saudi Pharm J 2018,26(8):1089-1097.
45.Miura M,Ito K,Hayashi M,Nakajima M,Tanaka T,Ogura S:The Effect of5-Aminolevulinic Acid on Cytochrome P450-Mediated Prodrug Activation.PLoS One2015,10(7):e0131793.
46.Ruettinger RT,Wen LP,Fulco AJ:Coding nucleotide,5'regulatory,anddeduced amino acid sequences of P-450BM-3,a single peptide cytochrome P-450:NADPH-P-450 reductase from Bacillus megaterium.J Biol Chem 1989,264(19):10987-10995.
47.Kang JY,Kim SY,Kim D,Kim DH,Shin SM,Park SH,Kim KH,Jung HC,Pan JG,Joung YH et al:Characterization of diverse natural variants of CYP102A1 foundwithin a species of Bacillus megaterium.AMB Express 2011,1(1):1.
48.Werz O,Gerstmeier J,Libreros S,De la Rosa X,Werner M,Norris PC,Chiang N,Serhan CN:Human macrophages differentially produce specific resolvinor leukotriene signals that depend on bacterial pathogenicity.Nat Commun2018,9(1):59.
序列表
<110> 赢创运营有限公司
<120> 包含巨大芽孢杆菌属益生菌菌株和多不饱和脂肪酸组分的制剂
<130> 文件编号: 201800212
<160> 37
<170> PatentIn version 3.5
<210> 1
<211> 1549
<212> DNA
<213> 巨大芽孢杆菌（Bacillus megaterium）DSM 32963
<400> 1
tcggagagtt tgatcctggc tcaggatgaa cgctggcggc gtgcctaata catgcaagtc 60
gagcgaactg attagaagct tgcttctatg acgttagcgg cggacgggtg agtaacacgt 120
gggcaacctg cctgtaagac tgggataact tcgggaaacc gaagctaata ccggatagga 180
tcttctcctt catgggagat gattgaaaga tggtttcggc tatcacttac agatgggccc 240
gcggtgcatt agctagttgg tgaggtaacg gctcaccaag gcaacgatgc atagccgacc 300
tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac gggaggcagc 360
agtagggaat cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa 420
ggctttcggg tcgtaaaact ctgttgttag ggaagaacaa gtacaagagt aactgcttgt 480
accttgacgg tacctaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 540
cgtaggtggc aagcgttatc cggaattatt gggcgtaaag cgcgcgcagg cggtttctta 600
agtctgatgt gaaagcccac ggctcaaccg tggagggtca ttggaaactg gggaacttga 660
gtgcagaaga gaaaagcgga attccacgtg tagcggtgaa atgcgtagag atgtggagga 720
acaccagtgg cgaaggcggc tttttggtct gtaactgacg ctgaggcgcg aaagcgtggg 780
gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta 840
gagggtttcc gccctttagt gctgcagcta acgcattaag cactccgcct ggggagtacg 900
gtcgcaagac tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 960
tttaattcga agcaacgcga agaaccttac caggtcttga catcctctga caactctaga 1020
gatagagcgt tccccttcgg gggacagagt gacaggtggt gcatggttgt cgtcagctcg 1080
tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt agttgccagc 1140
attcagttgg gcactctaag gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg 1200
tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaaag 1260
ggctgcaaga ccgcgaggtc aagccaatcc cataaaacca ttctcagttc ggattgtagg 1320
ctgcaactcg cctacatgaa gctggaatcg ctagtaatcg cggatcagca tgccgcggtg 1380
aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg taacacccga 1440
agtcggtgga gtaaccgtaa ggagctagcc gcctaaggtg ggacagatga ttggggtgaa 1500
gtcgtaacaa ggtagccgta tcggaaggtg cggctggatc acctccttt 1549
<210> 2
<211> 1549
<212> DNA
<213> 巨大芽孢杆菌DSM 32963
<400> 2
tcggagagtt tgatcctggc tcaggatgaa cgctggcggc gtgcctaata catgcaagtc 60
gagcgaactg attagaagct tgcttctatg acgttagcgg cggacgggtg agtaacacgt 120
gggcaacctg cctgtaagac tgggataact tcgggaaacc gaagctaata ccggatagga 180
tcttctcctt catgggagat gattgaaaga tggtttcggc tatcacttac agatgggccc 240
gcggtgcatt agctagttgg tgaggtaacg gctcaccaag gcaacgatgc atagccgacc 300
tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac gggaggcagc 360
agtagggaat cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa 420
ggctttcggg tcgtaaaact ctgttgttag ggaagaacaa gtacaagagt aactgcttgt 480
accttgacgg tacctaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 540
cgtaggtggc aagcgttatc cggaattatt gggcgtaaag cgcgcgcagg cggtttctta 600
agtctgatgt gaaagcccac ggctcaaccg tggagggtca ttggaaactg gggaacttga 660
gtgcagaaga gaaaagcgga attccacgtg tagcggtgaa atgcgtagag atgtggagga 720
acaccagtgg cgaaggcggc tttttggtct gtaactgacg ctgaggcgcg aaagcgtggg 780
gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta 840
gagggtttcc gccctttagt gctgcagcta acgcattaag cactccgcct ggggagtacg 900
gtcgcaagac tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 960
tttaattcga agcaacgcga agaaccttac caggtcttga catcctctga caactctaga 1020
gatagagcgt tccccttcgg gggacagagt gacaggtggt gcatggttgt cgtcagctcg 1080
tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt agttgccagc 1140
atttagttgg gcactctaag gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg 1200
tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaaag 1260
ggctgcaaga ccgcgaggtc aagccaatcc cataaaacca ttctcagttc ggattgtagg 1320
ctgcaactcg cctacatgaa gctggaatcg ctagtaatcg cggatcagca tgccgcggtg 1380
aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg taacacccga 1440
agtcggtgga gtaaccgtaa ggagctagcc gcctaaggtg ggacagatga ttggggtgaa 1500
gtcgtaacaa ggtagccgta tcggaaggtg cggctggatc acctccttt 1549
<210> 3
<211> 1188
<212> DNA
<213> 巨大芽孢杆菌DSM 32963
<400> 3
atgaagaatg gatggacaaa ctttgttatt ggtaccgtac gtattcgaat tgttggaaaa 60
ggaattgaac gttttttaaa taattgtgtg agacaacaga ttatgatttc gaatgtccat 120
aaagttgacg gtcaactagc aacagcaacg atattattga aagatgtaaa gaaaattcgt 180
atactaattc gcaacgcaga ttgtaaaatt tattttataa gaggcagagg ttttcctttt 240
ttaacaaaac gagtgatcaa aaatagtgga tttgctttag gttttctttc tttttttatt 300
attcttggtt tgctttccaa catggtgtgg aaagtggaaa ttagcggtgc agaaccacaa 360
acagaacatc aaatgactaa gcagcttgcc aaaataggtg taaaaagagg agagtttcaa 420
tttctgttag aaagtcctga aaaaattcag cgctatttaa cggataatat gaacaatatt 480
acctgggtgg gagtagaagt gcgcggcact tcttatcact tccaagtggt tgaaaaaaat 540
gagcccaagc ctcagcaaaa aacaccttac caacatttga ttgctaaaaa gaaagccatt 600
attacgaatt tgtttgtcga aaaaggacaa cctttagtca aggttaatga ttttgtcaat 660
gaaggcgagg tcctagtttc cggtattatt ggaaacgaaa aaaataaaaa ggtagtagct 720
gctaaaggga aagtctacgg tgaaacatgg tataagtctg aggtggaagt gcctttaaaa 780
actgattttc aggtattaac gggtaatgga tatacgaagc actatttaga ttttcaagct 840
tttaaaatgc ctctttgggc ttttaacaaa gaaaaatacg cctcaaaggt gacagaaaaa 900
gtggagcatc cactctattt tttaaaatgg aaattacctc tgtcgtatga gaaagttgct 960
gtacgagaag aacaaaactc gcagcgcgtc tattctaaac aagaggcggt ggaaaaagct 1020
ttggaaatcg gtcgaaaaaa attactatca actttaggtg aggattctaa aataaaaggt 1080
gaaaaagttt tgcaccaaga acaggacaat ggtaaagtta gattatcaat acattaccaa 1140
gttatagaaa atattgcgaa cactcaacca attattcaag gagattga 1188
<210> 4
<211> 1932
<212> DNA
<213> 巨大芽孢杆菌DSM 32963
<400> 4
gtgatacatt tgacgatgga acaaaaagaa gtacaagcat atgaagctga tcagatacaa 60
gtattagaag gattagaggc tgttcgtaaa cgtccgggga tgtatattgg atcgacgagc 120
gcaaagggtt tacatcatct tgtatgggaa attgtagata atagtattga tgaagcgctg 180
gccggctatt gcgatgaaat taatgttatt atcgaaaagg ataatagtat tacagtcaaa 240
gataacggtc gtggaattcc ggttggtatt caagaaaaaa tgggcagacc tgccgttgaa 300
gttatcttaa cggttcttca tgccggaggt aaatttggcg gcggcggcta taaagtatcc 360
ggtggattac atggtgtagg tgcctcagtt gttaacgcac tttctacctc tttggaagtg 420
tacgtacatc gtgatggtaa agttcattat caaaaatatg aacgaggtgt accggctgct 480
gacttaaaag tagttggaga aacagataaa acaggtactg ttattcaatt ccatccagac 540
ggtgaaattt ttacagaaac gcttgaatac gattttgata cgttagctaa tcgtctgcgt 600
gagttagctt tcttaaatcg cggtattaaa attacgattg aagacaaacg tgaagaagat 660
aaaagacgtg agtatcacta tgaaggcgga attaagtctt acgttgaaca cttaaaccgt 720
gcaaaagaag taattcacga agagccgatc tatattgaag gtaatcgaga caacatttct 780
gtagaaattg ctattcaata taacgatagc tatacaagta atttatattc ttttgcaaac 840
aacattcaca catatgaagg tggaacgcac gaagcaggat ttaaaacagc gttaacgcgt 900
gtaattaacg actatgcacg taaaaacagc gtatttaaag acagtgatgc caatctaacg 960
ggtgaagatg ttcgtgaagg aattacagct atcatctcta ttaagcaccc agatccgcag 1020
tttgaaggac aaacaaaaac aaagctggga aatagtgaag caagaacaat tactgactct 1080
gtgtttgcag aacacttaga aacgtacttg ctagagaacc ctattgtggc gaaaaaggta 1140
attgaaaaag gtttaatggc tgcaagagca agaatggcag ctaaaaaagc tcgtgagctt 1200
acaagacgta aaagcgcgct tgaaatctca aacttaccgg gtaaattagc agattgttca 1260
tcaaaagatc cttctattag cgaactctat gtagtagagg gtgactctgc gggaggatca 1320
gctaagcagg gaagaagccg tcatttccaa gctattttgc ctttacgtgg taaaattatc 1380
aacgtagaga aagcccgttt agataaaatt ttatctaata acgaaattcg tacaatcatt 1440
accgctctag gaacgggtat tggtgacgat tttgatatct cgaaagcacg ctaccataaa 1500
attgtgatca tgacagatgc agacgtggac ggtgcgcata ttcgtacgct tcttctaacg 1560
ttcttctatc gctatatgag acagattatt gagcatggat atgtgtacat tgcccagccg 1620
cctctttaca aagtttcaca aggtaaaaaa gtggagtacg cgtacaacga tcgtcaatta 1680
gaagaggtat tagcttcttt ccctgaaggc gcaaaaccaa accttcagcg ttacaaaggt 1740
ttaggagaga tgaatcctga acaattatgg gaaacaacaa tggatccaga gttccgtacc 1800
cttcttcaag tgaacttgca agatgcaatt gaagctgatg agacatttga aattttaatg 1860
ggcgacaaag tagaaccacg ccgtaatttc attgaagaaa atgctcagta cgtaaaaaat 1920
cttgatattt aa 1932
<210> 5
<211> 3573
<212> DNA
<213> 巨大芽孢杆菌DSM 32963
<400> 5
gtgaatcagt tgacaggtca actagttcag tatggacgcc accgccaacg cagaagttat 60
gctcgtatta gtgaagtttt agagttaccg aacttaatcg agattcaaac ggcttcatac 120
caatggtttt tagatgaagg tttacgagaa atgttccaag atatttctcc gattgatgat 180
tttacaggta acttatcact agaatttatt gattacagct taggtgagcc aaagtattca 240
gtaggagagt ccaaagaacg tgatgtaact tatgcagcac ctcttcgtgt taaggtgcgg 300
ttaattaata aagaaacagg tgaagtaaaa gaccaagatg tgttcatggg agatttccca 360
ttgatgaccg aaacaggtac ctttgtgatt aatggtgctg agcgtgttat cgtatcacag 420
ttggttcgtt caccaagtgt ttactatagt ggaaagcttg ataaaaacgg taaaaaagga 480
tatacagcaa ctgttatccc taaccgtggt gcatggctag aatatgaaac tgatgcaaaa 540
gatgtagtat acgtgcgtat tgatcgtact cgtaaactgc cagtaacagt gctgttacgc 600
gcgctaggtt tcggttctga ccaagagatt atcgatttaa tcggtgataa tgaatacatc 660
cgtaatacgc ttgaaaaaga taatacggaa acaacggaaa aagcgctatt agaaatctat 720
gagcgtttac gtccgggtga accaccgaca gttgagaatg cgaagtctct attagtatct 780
cgcttctttg atccaaaacg atatgactta gcgaacgtag gtcgctataa aattaataaa 840
aagcttcata tcaaacatcg cttatttaat caaaagctag ctgaaacatt agttgatcct 900
gaaacaggtg aaattattgc agaaaaaggc gcaatgattg accgccgtct gctagatcgc 960
ttgattccga tgcttgaagg tggagtaaac ttcaaaactt atagtccggt tggtggagta 1020
gtagaagacg atgttacatt acaatctatt aagatttatg cgccaaatga tccagaaggt 1080
gaaaaaatca tcactgtatc aggtaacgca tatgtaacag aagaagttaa aaatatcaca 1140
cctgctgata ttttagcatc aattagttac ttctttaact tgcttcatca agtaggagac 1200
acagatgata tcgaccactt aggtaaccgt cgtctgcgtt ctgtaggtga attgttacaa 1260
aaccaattcc gtatcggttt atctcgtatg gaacgtgttg ttcgtgaaag aatgtcaatt 1320
caagacacga atacaatcac acctcaacaa ttaattaata ttcgcccagt tattgcggcg 1380
attaaagagt tctttggaag ttctcaatta tcacagttca tggatcaaac gaatccatta 1440
ggcgaattga cgcacaaacg tcgtctttca gctctaggac ctggtggttt aacgcgtgag 1500
cgcgctggtt ttgaagtgcg tgacgttcac tactcccact atggccgtat gtgtccgatt 1560
gaaactccag agggtccgaa tatcgggtta atcaactcac tatcttctta tgcaaaagta 1620
aacaaattcg gtttcatcga aacaccttac cgtcgtatcg atcctgaaac aggtaaagtg 1680
acagagcgaa ttgactactt aacagctgat gaagaagata actatgttgt agcccaagcg 1740
aacgctcgtc taggcgatga tggttcattc ttagatgaaa atgtcgttgc acgtttccgt 1800
ggagaaaaca cggttatccg tcgcgatcgt ttagattata tggatgtatc accaaaacaa 1860
gttgtatctg ccgctacagc atgtatccca ttcttagaga acgatgactc taaccgtgca 1920
ttaatgggtg cgaacatgca acgtcaagca gtacctttat taaatcctga agcaccaatc 1980
gtaggtacag gtatggaata cgtatctggt aaagactctg gtgcagccgt gatttgtaaa 2040
tatcctggcg ttgtagagcg cgtagaagca aaacaaattt ttgttcgccg ctatgaagaa 2100
gtagacggac aaaaagttaa aggtaactta gatcaataca aattattaaa atttgttcgt 2160
tctaaccaag gtacttgtta caaccagcgt ccaattgttt cagttggcga cgaagtagta 2220
aaaggtgaga tcttagccga cggtccttca atggaaaaag gtgagcttgc tttaggacga 2280
aacgtaatgg ttggtttcat gacatgggat ggttacaact atgaggatgc catcatcatg 2340
agtgaacgcc ttgtgaaaga tgatgtatat acgtctgttc atattgaaga atatgaatct 2400
gagtctcgtg atacgaagct tggacctgaa gaaattacgc gtgacattcc aaacgtaggt 2460
gaagatgcgc ttcgcaactt agatgagcgt ggaatcatcc gcattggtgc agaagtaaaa 2520
gacggagatc ttttagttgg taaagtaacg ccaaaaggtg taacagaact aacagctgaa 2580
gaacgtcttc tacacgctat tttcggtgaa aaagcgcgtg aagttcgtga tacttctctt 2640
cgtgtaccgc acggcggcgg tggaatcatt cttgatgtta aagtcttcaa ccgtgaagat 2700
ggcgacgaat taccaccagg tgtaaaccaa ttagtccgtg tatatattgt tcagaagcgt 2760
aaaatttctg aaggtgacaa aatggccggt cgtcacggta acaaaggtgt aatttcacgt 2820
attttacctg aagaagatat gccttatcta ccagacggta cgccaattga catcatgtta 2880
aacccattag gggtaccatc tcgtatgaac atcggtcagg tgctagagct tcatttaggt 2940
atggctgctc gtaagcttgg cattcacgtt gcgtctccag tatttgatgg tgcgcgtgag 3000
gaagatgtgt gggcaacaat cgaagaagct ggcatgtctc gtgatgctaa aacagttcta 3060
tatgatggtc gaacaggtga accattcgat aaccgtgtat cagtaggaat catgtacatg 3120
atcaaacttg ctcacatggt agacgataaa cttcacgctc gttctactgg accatactca 3180
cttgttacac aacaaccact tggtggtaaa gcgcagttcg gtggacagcg ttttggtgag 3240
atggaggtat gggcacttga agcatacggt gctgcttaca cattacaaga gatcttaaca 3300
gtgaaatcag atgacgtagt aggtcgtgtg aaaacatacg aagcaattgt taaaggtgaa 3360
aacattccag aacctggcat acctgaatcg ttcaaagtat taattaaaga actacaaagt 3420
ttaggtatgg atgtgaagat gctttctgct gacgagcaag aaattgatat catggactca 3480
gaagaggacc atgagcaacc aacagaatca attattgctg ataacgaaga aagcctttct 3540
gaaggacaaa aagatcctgt cacaaaagag taa 3573
<210> 6
<211> 1632
<212> DNA
<213> 巨大芽孢杆菌DSM 32963
<400> 6
atggcaaaag acattaaatt tagcgaagaa gcacgtcgcg caatgctacg tggtgtagac 60
acattggcaa atgctgtaaa agtaacgctt ggaccaaaag gtcgtaacgt tgtattagaa 120
aagaaattcg gttcaccgct tattacaaat gacggtgtaa caattgcaaa agaaatcgaa 180
ttagaagacg catttgaaaa tatgggtgct aaattagtag ccgaagttgc tagcaaaaca 240
aacgacgtcg ctggtgacgg tacaactact gcaacagttt tagcgcaagc aatgatcaga 300
gaaggtctta aaaacgtaac ggctggtgct aacccaatgg gtatccgtaa aggtatggaa 360
aaagcagtag ctgtagcggt tgaagaacta aaagcaatct ctaaaccaat tcaaggtaaa 420
gattcaattg ctcaagtagc ggctatctca gcagctgacg aagaagtagg tcaattaatt 480
gctgaagcaa tggagcgcgt tggtaacgac ggcgttatca cacttgaaga atcaaaaggt 540
ttcacaactg aattagaagt ggtagaaggt atgcagtttg accgtggata tgcatctcct 600
tacatggtaa ctgattcaga taaaatggaa gctgtattag atgatccata catcttaatc 660
acagacaaaa aaatcggtaa cattcaagaa atcttaccgg tattagagca agttgttcaa 720
caaggcaagc ccctattaat catcgctgaa gacgtagaag gcgaagcttt agcaacatta 780
gttgtgaaca aacttcgtgg tacattcaca gctgtagctg ttaaagctcc tggtttcggt 840
gatcgtcgta aagcaatgct acaagacgtt gcgatcttaa caggcggaga agtaatcact 900
gaagagcttg gtctcgacct aaaaacagca agcattgatc aattaggtcg cgcttctaaa 960
attgttgtaa caaaagaaaa tacaacggtt gtaaacggtg caggaaacgc agaagacatc 1020
ctagcacgcg taaaccaaat caaagctcag cttgaagaaa caacttcaga gtttgaccgt 1080
gaaaaattac aagagcgctt agcaaaactt gctggtggcg tagctgtaat taaagtgggt 1140
gcggcaactg aaactgagtt aaaagaacgt aaattacgta ttgaagatgc attaaactct 1200
acgcgtgctg cggttgaaga aggtatcgta gctggtggtg gtactgcatt agtaaatatc 1260
tataataaag tagcaagcat cgaagctgac ggtgacactg ctacaggtat caacattgta 1320
ttacgtgcga tcgaagagcc tgtacgtcaa atcgctcaca atgctggttt agaaggatca 1380
gtaatcgttg agcgtctaaa aggcgaagca gttggaactg gattcaatgc tgcaactggc 1440
gagtgggtaa acatgctaga cactggtatc gttgacccaa caaaagtaac gcgttcagct 1500
cttcaaaatg cttcttctgt agcggctatg ttcttaacaa ctgaagcagt tgttgctgac 1560
aagccagaag aaggcggagc acctgcaatg cctgacatgg gcggcatggg tggaatgggc 1620
ggcatgatgt aa 1632
<210> 7
<211> 3150
<212> DNA
<213> 巨大芽孢杆菌DSM 32963
<400> 7
atgacaatta aagaaatgcc tcagccaaaa acgtttggag agcttaaaaa tttaccgtta 60
ttaaacacag ataaaccggt tcaagctttg atgaaaattg cggatgaatt aggagaaatc 120
tttaaattcg aggcgcctgg tcgtgtaacg cgctacttat caagtcagcg tctaattaaa 180
gaagcatgcg atgaatcacg ctttgataaa aacttaagtc aagcgcttaa atttgtacgt 240
gattttgcag gagacgggtt atttacaagc tggacgcacg aaaaaaattg gaaaaaagcg 300
cataatatct tacttccaag cttcagtcag caggcaatga aaggctatca tgcgatgatg 360
gtcgatatcg ccgtgcagct tgttcaaaag tgggagcgtc taaatgcaga tgagcatatt 420
gaagtaccgg aagacatgac acgtttaacg cttgatacaa ttggtctttg cggctttaac 480
tatcgcttta acagctttta ccgagatcag cctcatccat ttattacaag tatggtccgt 540
gcactggatg aagcaatgaa caagctgcag cgagcaaatc cagacgaccc agcttatgat 600
gaaaacaagc gccagtttca agaagatatc aaggtgatga acgacctagt agataaaatt 660
attgcagatc gcaaagcaag cggtgaacaa agcgatgatt tattaacgcg tatgctaaac 720
ggaaaagatc cagaaacggg tgagccgctt gatgacgaga acattcgcta tcaaattatt 780
acattcttaa ttgcgggaca cgaaacaaca agcggtcttt tatcatttgc gctgtatttc 840
ttagtgaaaa atccacatgt attacaaaaa gcagcagaag aagcagcacg agttctagta 900
gatcctgttc caagctacaa acaagtcaaa cagcttaaat atgtcggcat ggtcttaaac 960
gaagcgctgc gcttatggcc aactgctcct gcgttttccc tatatgcaaa agaagatacg 1020
gtgcttggag gagaatatcc tttagaaaaa ggcgacgaac taatggttct gattcctcag 1080
cttcaccgtg ataaaacaat ttggggagac gatgtagaag agttccgccc agagcgtttt 1140
gaaaacccaa gcgcaattcc gcagcatgcg tttaaaccat ttggaaacgg gcagcgcgcg 1200
tgtatcggtc agcagttcgc tcttcatgaa gcaacgcttg tacttggtat gatgctaaaa 1260
cactttgatt ttgaagatca tacaaactac gagctggatg ttaaagaaac tttaacgtta 1320
aaacctgaag gctttgtggt aaaagcaaaa tcgaaacaaa ttccgcttgg cggtattcct 1380
tcacctagca ctgaacagtc tgttaaaaaa gtacgcaaaa aggtagaaaa cgctcataat 1440
acgccgctgc ttgtgctata cggttcgaac atgggaacag ccgaaggaac ggcgcgtgat 1500
ttagcggata ttgcgatgag caaaggattc gcaccgcagg tcgcagcgct tgattcccac 1560
gcaggaaatc ttccgcgcga aggagctgta ttaattgtaa cggcgtctta taacggacac 1620
ccgcctgata acgcaaagca atttgtcgac tggttagacc aagcgtctgc tgatgaagta 1680
aaaggcgttc gctactccgt atttggatgc ggcgataaaa actgggctac tacgtatcaa 1740
aaagtgcctg cttttattga tgaaacgctt gccgctaaag gagcagaaaa catcgctgaa 1800
cgcggtgaag cagatgcaag cgacgacttt gaaggcacat atgaagaatg gcgtgaacac 1860
atgtggagtg acgtagcagc ctactttaac ctcgacattg aaaacagtga agataataaa 1920
tctacgcttt cacttcaatt tgtcgacagc gctgcggaca tgccgcttgc taaaatgcac 1980
ggtgcgtttt cagcaaacgt cgtagcaagc aaagaacttc aacagccagg cagtgaacga 2040
agcacgcgtc atcttgaaat tgaacttcca aaagaaactt cttatcaaga aggagatcat 2100
ttaggcgtta ttcctcgcaa ctatgaagga atagtaaatc gtgtgacgac aagatttggt 2160
ctagatgtat cacagcaaat ccgcttggaa gcagaagaag aaaaattagc gcatttgcca 2220
ctcggtaaaa cagtatcagt agaagagctt ctgcaatacg tagagcttca agaccctgtt 2280
acgcgcacgc agcttcgcgc aatggctgct aaaacggtct gcccgccgca taaagtagag 2340
cttgaagcct tgcttgaaaa gcaagcctac aaagaacaag tgctggcaaa acgtttaaca 2400
atgcttgaac tgcttgaaaa atacccggcg tgtgaaatgg aattcagcga atttatcgcc 2460
cttctgccaa gcatacgccc gcgctattat tcaatttcat cttcacctcg tgtcgatgaa 2520
aaacaagcaa gcattacggt cagcgttgtc tcaggagaag cgtggagcgg atacggagaa 2580
tacaaaggaa ttgcgtcaaa ctaccttgcc gagctgcaag aaggagatac gattacgtgc 2640
tttgtttcta caccgcagtc aggatttacg ctgccaaaag accctaaaac gccgcttatc 2700
atggtcggac cgggaacagg cgttgcaccg tttagaggct ttgtgcaggc tcgcaagcag 2760
ctaaaagaac aagaagagtc gcttggagaa gcgcatttat actttggctg ccgttcacct 2820
catgaagact atctgtatca agaagagctt gaaaacgccc aaaatgaagg catcattacg 2880
cttcataccg ctttttctcg cgtaccaaat cagccgaaaa catacgttca gcacgtaatg 2940
gaacaagacg gcaagaaatt gattgaactt cttgaccaag gagcgcactt ctatatttgc 3000
ggagacggaa gccaaatggc acctgacgtt gaagcaacgc ttatgaaaag ctatgctgac 3060
gttcaccaag tgagtgaagc agacgctcgc ttatggctgc agcagctaga agaaaagggc 3120
cgatacgcaa aagacgtgtg ggctgggtaa 3150
<210> 8
<211> 1812
<212> DNA
<213> 巨大芽孢杆菌DSM 32963
<400> 8
gtgttgcaac ttaaggtagt aaacagccct tttaatcaag aacaagcaga tttgcttaat 60
cgccttctgc cgacattaac agaagcacaa aaaatgtggt tgagcggtta tttaacagca 120
gctcaatcta cgtctgccga aggaacgcca gacgtttcta cagcagcgcc tgctcaaacg 180
aaacagacag tttcaaaaga cgtaacgatt ctttatggat cacaaacagg aaatgctcaa 240
ggacttgctg aaaatacagg caaaacgctt gaagcaaaag gctttaatgt aactgtatct 300
tctatgaatg atttcaaacc aaataattta aagaaacttg aaaatttatt aattgtcgta 360
agtacacatg gagaaggaga gccgcctgat aatgcgctat ctttccatga atttcttcac 420
ggccgtcgag cgccaaaact tgagaacttc cgtttctctg ttttatcgct tggagacagc 480
tcatacgaat ttttctgtca aacaggaaaa gaatttgatg tgcgcttagc agaacttggc 540
ggtgaaagac tgtatccgcg cgttgactgt gatttagatt ttgaagagcc cgcaaataaa 600
tggcttaaag gtgttattga cggattaagc gaagcgaaag gacacagcgc ttcggcagct 660
gttccagcgg aagctcctgc aggaacttca ccatattcaa gaacaaaccc ttttaaagca 720
gaagtgcttg agaacttaaa cttaaacggc cgcggatcaa ataaagaaac gcgacactta 780
gaactatctc tagaaggttc aggtttgacg tatgaaccag gagacagttt aggtatttat 840
cctgaaaatg acccggagct tgttgatctt cttcttaacg aattcaagtg ggatgcaagt 900
gaaagtgtaa cggttaataa agaaggagaa acgcgtcctc ttagagaagc gctaatctct 960
aattttgaaa ttaccgtttt aacaaagccg cttttaaagc aagcagctga gcttactgga 1020
aacgataagt taaaagcgct tgtagaaaat cgcgaggaat taaaagcata cacacaaggc 1080
cgtgatgtaa ttgacttagt tcgtgacttc ggtccatgga acgtatcggc acaagagttt 1140
gtagccattc tacgtaaaat gccagcgcgt ctttactcga ttgcaagcag cttatcagca 1200
aaccctgatg aagttcatct aacaatcgga gcggtacgct acgaagcgca tggacgcgag 1260
cgtaaaggtg tttgttcagt cctttgttca gaacgtttgc agccaggcga tacgattcct 1320
gtataccttc aaagcaataa aaactttaag cttcctcaag atcaagaaac gccaattatt 1380
atggtaggac ctggtacagg tgtggctccg ttccgctcat ttatgcaaga gcgtgaagaa 1440
acaggggcaa aaggaaagtc atggatgttc tttggagatc agcacttcgt aacagacttc 1500
ctttaccaaa cagaatggca aaagtggtta aaagatggcg tactgacaaa aatggacgtg 1560
gcgttttcac gcgatacaga agaaaaagta tacgtacaaa accgtatgct cgaacatagt 1620
aaagaattat tccaatggtt agaagaaggc gcatcttttt atgtgtgcgg agataaaaca 1680
aatatggcac gcgacgtgca caacacgcta gttgaaatta tcgaaacaga aggcaagatg 1740
agccgtgaac aggcggaagg ttaccttgct gaaatgaaga aacaaaaacg ttatcagcgt 1800
gatgtatact ga 1812
<210> 9
<211> 1278
<212> DNA
<213> 巨大芽孢杆菌DSM 32963
<400> 9
ttgccggtta tacagggccc ctcgtcgtat aagcttaccg gtcacctgca aaagtttaga 60
gagaaccccc tgggtttttt ggaaaacttg actcagtatg gagaaattgc aacatttaga 120
gttgcgcaca aacgctttta tgtaacgaga gaccctcaac ttattaaaga tgtagtcatt 180
acaaacagca aggcgtttca aaaaattaaa ctaacgcata tgtttaaaac gctgctaggc 240
gaagaaatgt tatggacaga tgaagcatta tatatgagcc ccattcaacc ctcacaacta 300
aaacaacatc taacttttaa taaggaagca atcgcaaaaa taattgaaaa gcatacggaa 360
acgtgggaag aagggcagct tcgaacaata gtaaaagaca taagacagat agtcatagct 420
gtactccttc aacttgtatt tggcatttcg attgaggaga aagataaaat tcactatgtt 480
caagcgctta tgagaaaaaa agaaaagtta ggcaaaattt atattcgtct acctttgcac 540
cagcctgact cagatgagca gctagaacaa ctcctttttg aacgcgttca aatgcgtgtt 600
caaaacaaaa ccgcaggaaa tgatttactg cagtatatat tgaattctta cggagaagac 660
agcgatgaaa gagaaatata cgaacagcta aattctatat tcctttcgat gtatgaaatg 720
attacgcacg tatgcagctg gtctattcat ttactatctc aaaatactag ggagcacctt 780
cagctgcata aagaaattca agcctatgct agcggcgaat cactttcaac caaaaatctg 840
acctatatgc gcaaaataat tgctgaaagc atgaggttgt atccgcctct ttggctattt 900
gggcgtcaag cacgtgaaga tatacaaata gatggctaca gtattaagaa aggggagatt 960
atgttaatta gcccttatat gatgcatcgc catgaagatt attttttaga gccaagtgaa 1020
tttttacctg accgttttga aaaaggaggg tccattgatg tcccaagcta tatgtatatg 1080
ccacttggga ttgagcatca agcggaaagg ggcatggact atatcactga aatagtcacc 1140
atttttctgt cagaaatgac aaagcgcttt ttatttcagc tgactaagcc cgaatctatt 1200
gctccgatgg cgggcgtaat gctgaatatg aaagaagaac tgaaggtgaa tgttcacaaa 1260
gttcacgcgc agtcttga 1278
<210> 10
<211> 1215
<212> DNA
<213> 巨大芽孢杆菌DSM 32963
<400> 10
atgaaaacag aaagagaaaa cggaattgtc cgtcaagtga atacgattca aacaaaagaa 60
gaacgcttta atcctttttc atggtatgaa gagatgagaa acaccgagcc tgtgcaatgg 120
gatgaaaaaa ggcaggtatg ggatgttttt cactatgacg gggtcaaaga agtgctagaa 180
caaaagaata ttttttcttc tgatcgaaga cctccacaaa accaaagaca aactgctcta 240
ggaacgagcc taattaatat tgatccgcct aagcatgctg aaatgagagc gcttgttaat 300
aaagctttta cgcctaaagc aatgaaagca tgggagccta aaattgcgcg tattacaaat 360
gaattattac aagaagttga gcatcttgaa gatattgata tagtcgagca tctgtcctat 420
ccgcttccgg ttatggtaat tgccgatatt ttaggggtgc cgatagaaga tcagcgtcag 480
tttaaagatt ggtcggatat tatcgtagcc ggtccttcta ataacgaacg tgaaacgctc 540
gaaaaactcc agcaagagaa aatgaaagca aatgatgagc tagaaactta cttttatcga 600
atcattgaag aaaaacgcac gcgtccagga gatgatatta tctccctgct tcttcaggca 660
aaagaagaag ggaaacagct aacggatgaa gagatcgtcg gtttttctat tttgctactg 720
attgctggaa acgaaacaac cacaaattta atttcaaata cgatttattg tttaatggaa 780
gataaagctt cttttgaacg actcaaacga gagaaagaac ttttgccttc tgcgattgaa 840
gaagttcttc gctatcgttc acccgttcag gctcttcacc gaatcgtaaa agaagatgtg 900
actcttgcag gcaagaaact aaaagcgggc gaacacgtcg ttccatggat gggatctgcg 960
catcgagacg cggagtattt tgaagatccg gatgtattta aaatcgatcg aaagccaaat 1020
gtgcacatgg catttggaag aggcattcat ttttgcttag gagcacccct tgctcgaata 1080
gaagcaaaaa ttatgctggc cgagctgatt gaccgctatc cgcagatgga ctggagcccg 1140
tcatttgagt taaagccaat tgaaagcacg tttgtgtacg ggttaaaaga attattgatt 1200
cgtaaaaacg tataa 1215
<210> 11
<211> 1212
<212> DNA
<213> 巨大芽孢杆菌DSM 32963
<400> 11
atgaatccaa aagcagtgaa aagagaaaat cgttatgcta atttaattcc tatgcaagaa 60
attaaatctg ttgaacagca gctttaccca tttgatattt ataatagcct gcgtcaagag 120
gcacctattc gttatgatga aagccgaaac tgctgggatg tatttgatta tgaaacagta 180
aagtatattt taaaaaaccc gtcgcttttt tcatccaaac gagcaatgga agaacgccaa 240
gaaagtattt taatgatgga tcctccaaaa catacaaagt tacgaaattt agtaaacaaa 300
gcttttacgc caagagctat tcagcacttg gagggtcata ttgaagagat tgcagattat 360
ttattagatg aagtaagcag caaagaaaag tttgatatag tagaagactt tgctggacca 420
ctgccgatta ttgtaatagc agagctgcta ggagtaccaa tacaagaccg agcactgttt 480
aaaaagtact ccgatgactt agtgagcggt gccgaaaata attcagacga agcttttgcg 540
aaaatgatgc aaaagcgtaa tgaaggcgtg atttttttac aagggtattt taaagaaatt 600
atcgcggagc gtcagcaaaa taaacaagaa gatctcattt cgcttctttt agaagcagaa 660
atcgacggcg agcatttaac agaagaggaa gtgctggggt tttgtatttt attgctagta 720
gcaggtaatg agacaacaac aaacttaatt acgaacggag ttcggtatat gacagaagat 780
gtagacgtgc agaatgaagt tcgtcgagac atatcgctag ttccgaacct ggtagaagaa 840
acgcttcgct attatcctcc tattcaagcg attggacgca tagctactga agatgtagaa 900
cttggagagt gtaagattaa aagaggacag caggtaatta gctgggcagc gagtgcaaat 960
agagattcag ctaagtttga atggcctgac acttttgtcg tgcatcgtaa aacaaatcct 1020
cacgtgagct ttggatttgg catccatttt tgcttaggag caccactagc aagaatggaa 1080
gggaaaattg cttttactaa gttactagaa aaagggggat tttccaaagt tcagaatcag 1140
tctttaaagc caatagatag cccttttgtt tttggtgtaa aaaaatatga aattgctttt 1200
aataacgcct ga 1212
<210> 12
<211> 1233
<212> DNA
<213> 巨大芽孢杆菌DSM 32963
<400> 12
atgaacaaag aagtcattcc cgttacagaa attccaaaat ttcaatcacg tgcagaagag 60
tttttcccta tccaatggta taaagaaatg ttgaacaact cccccgttta ttttcatgaa 120
gaaacaaaca catggaatgt attccaatac gaacatgtta aacaagtctt gagcaactac 180
gatttttttt caagcgatgg acagagaacc actatttttg ttggagataa tagtaagaaa 240
aaaagcacat ctccaatcac caaccttaca aacttagatc ctcctgacca ccggaaagcg 300
cgatctttgc ttgctgcagc ctttactcct cgcagtttaa ggaactggga accgcgaatc 360
aaacaaattg cagcagaact tgtagaagcg atacaaaaga attcaactat taatattgtg 420
gacgatttat cttctccttt tcctagtcta gttatagcag atctattcgg cgtaccggta 480
aaagaccgat atcagtttaa aaaatgggta gacattctgt ttcagcctta tgatcaagaa 540
agattagaag agattgaaca agaaaagcaa cgtgcaggag ctgaatattt tcaatacctc 600
tacccgattg tcattgaaaa gcgctccaac ctttctgatg atattatctc agacttaatt 660
caagctgaag ttgatggtga aacattcaca gataaagaaa ttgtacacgc taccatgttg 720
cttctaggtg caggcgttga aaccacaagt cacgccattg caaatatgtt ttattctttt 780
ttatatgacg acaagtcgtt atacagcgag ctgagaaaca atagagaatt agctccaaaa 840
gcagtagaag aaatgcttcg atatcggttt catatctcca gacgagatcg cacggttaaa 900
caagataacg agctattagg cgttaagcta aaaaaaggag atgtcgttat tgcgtggatg 960
agtgcatgta atatggatga aactatgttt gaaaaccctt tttctgtaga cattcatcgt 1020
ccaactaata aaaagcactt aacattcgga aacggacctc atttttgttt gggcgcccct 1080
cttgcccgac ttgaaatgaa aattatctta gaagcatttt tagaggcctt ttctcatatt 1140
gaaccatttg aagactttga attagaaccg catttaacag cttcagctac gggtcaatct 1200
ttaacatact tgcccatgac ggtgtacaaa taa 1233
<210> 13
<211> 1549
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33296
<400> 13
tcggagagtt tgatcctggc tcaggatgaa cgctggcggc gtgcctaata catgcaagtc 60
gagcgaactg attagaagct tgcttctatg acgttagcgg cggacgggtg agtaacacgt 120
gggcaacctg cctgtaagac tgggataact tcgggaaacc gaagctaata ccggatagga 180
tcttctcctt catgggagat gattgaaaga tggtttcggc tatcacttac agatgggccc 240
gcggtgcatt agctagttgg tgaggtaacg gctcaccaag gccacgatgc atagccgacc 300
tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac gggaggcagc 360
agtagggaat cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa 420
ggctttcggg tcgtaaaact ctgttgttag ggaagaacaa gtacaagagt aactgcttgt 480
accttgacgg tacctaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 540
cgtaggtggc aagcgttatc cggaattatt gggcgtaaag cgcgcgcagg cggtttctta 600
agtctgatgt gaaagcccac ggctcaaccg tggagggtca ttggaaactg gggaacttga 660
gtgcagaaga gaaaagcgga attccacgtg tagcggtgaa atgcgtagag atgtggagga 720
acaccagtgg cgaaggcggc tttttggtct gtaactgacg ctgaggcgcg aaagcgtggg 780
gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta 840
gagggtttcc gccctttagt gctgcagcta acgcattaag cactccgcct ggggagtacg 900
gtcgcaagac tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 960
tttaattcga agcaacgcga agaaccttac caggtcttga catcctctga caactctaga 1020
gatagagcgt tccccttcgg gggacagagt gacaggtggt gcatggttgt cgtcagctcg 1080
tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt agttgccagc 1140
atttagttgg gcactctaag gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg 1200
tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaaag 1260
ggctgcaaga ccgcgaggtc aagccaatcc cataaaacca ttctcagttc ggattgtagg 1320
ctgcaactcg cctacatgaa gctggaatcg ctagtaatcg cggatcagca tgccgcggtg 1380
aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg taacacccga 1440
agtcggtgga gtaaccgtaa ggagctagcc gcctaaggtg ggacagatga ttggggtgaa 1500
gtcgtaacaa ggtagccgta tcggaaggtg cggctggatc acctccttt 1549
<210> 14
<211> 1549
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33296
<400> 14
tcggagagtt tgatcctggc tcaggatgaa cgctggcggc gtgcctaata catgcaagtc 60
gagcgaactg attagaagct tgcttctatg acgttagcgg cggacgggtg agtaacacgt 120
gggcaacctg cctgtaagac tgggataact tcgggaaacc gaagctaata ccggatagga 180
tcttctcctt catgggagat gattgaaaga tggtttcggc tatcacttac agatgggccc 240
gcggtgcatt agctagttgg tgaggtaacg gctcaccaag gccacgatgc atagccgacc 300
tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac gggaggcagc 360
agtagggaat cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa 420
ggctttcggg tcgtaaaact ctgttgttag ggaagaacaa gtacaagagt aactgcttgt 480
accttgacgg tacctaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 540
cgtaggtggc aagcgttatc cggaattatt gggcgtaaag cgcgcgcagg cggtttctta 600
agtctgatgt gaaagcccac ggctcaaccg tggagggtca ttggaaactg gggaacttga 660
gtgcagaaga gaaaagcgga attccacgtg tagcggtgaa atgcgtagag atgtggagga 720
acaccagtgg cgaaggcggc tttttggtct gtaactgacg ctgaggcgcg aaagcgtggg 780
gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta 840
gagggtttcc gccctttagt gctgcagcta acgcattaag cactccgcct ggggagtacg 900
gtcgcaagac tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 960
tttaattcga agcaacgcga agaaccttac caggtcttga catcctctga caactctaga 1020
gatagagcgt tccccttcgg gggacagagt gacaggtggt gcatggttgt cgtcagctcg 1080
tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt agttgccagc 1140
atttagttgg gcactctaag gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg 1200
tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaaag 1260
ggctgcaaga ccgcgaggtc aagccaatcc cataaaacca ttctcagttc ggattgtagg 1320
ctgcaactcg cctacatgaa gctggaatcg ctagtaatcg cggatcagca tgccgcggtg 1380
aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg taacacccga 1440
agtcggtgga gtaaccgtaa ggagctagcc gcctaaggtg ggacagatga ttggggtgaa 1500
gtcgtaacaa ggtagccgta tcggaaggtg cggctggatc acctccttt 1549
<210> 15
<211> 1188
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33296
<400> 15
atgaagaatg gatggacaaa ctttgttatt ggtaccgtac gtattcgaat tgttggaaaa 60
ggaattgaac gttttttaaa taattgtgtg agacaacaga ttatgatttc gaatgtccat 120
aaagttgacg gtcagctagc gacagcaacg atattattga aagatgtaaa aaaaattcgt 180
atactaattc gcaacgcaga ttgtaaaatt tattttataa gaggcagagg ttttcctttt 240
ttaacaaaac gagtaatcaa aaatagtgga tttgctttag gttttctttc tttttttatt 300
attcttggtt tgctttccaa catggtgtgg aaagtggaaa ttagcggtgc agaaccacaa 360
acagaacatc aaatgactaa gcagcttgcc aaaataggtg taaaaagagg agagtttcaa 420
tttctgttag agagtcctga aaaaattcag cgctatttaa cagataatat gaacaatatt 480
acctgggtgg gagtagaagt gcgcggcact tcttatcact tccaagtggt tgaaaaaaat 540
gagcccaagc ctcagcaaaa aacaccttac caacatttaa ttgctaaaaa gaaagccatt 600
attacgaatt tgtttgtcga aaaaggacaa cctttagtca aggttaatga ttttgtcaac 660
gaaggcgagg tcctagtttc cggtattatt ggaaacgaaa aaaataaaaa agtagtagct 720
gctaaaggga aagtctacgg tgaaacatgg tataagtctg aggtggaagt gcctttaaaa 780
actgattttc aggtattaac gggtaatgga tatacgaagc actatttaga ttttcaagct 840
tttaaaatgc ctctttgggc ttttaacaaa gaaaaatacg cctcaaaggt gacagaaaaa 900
gtggagcatc cactctattt ttttaaatgg aaattacctc tgtcgtatga gaaagttgct 960
gtacgagaag aacagaactc gcagcgcgtc tattctaaac aagaggcggt ggaaaaagct 1020
ttggaaatcg gtcgaaaaaa attactatca actttaggtg aggatgctaa aataaaaggt 1080
gaaaaagttt tgcaccaaga acaggacaat ggtaaagtta gattatcaat acattaccaa 1140
gttatagaaa atattgcgaa cactcaacca attattcaag gagattga 1188
<210> 16
<211> 1923
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33296
<400> 16
ttgacgatgg aacaaaaaga agtacaagca tatgaagctg atcagataca agtattagaa 60
ggattagaag ctgttcgtaa acgtccgggg atgtatattg gatcgacgag cgcaaagggt 120
ttacatcatc ttgtatggga aattgtagat aatagtattg atgaagcgct ggccggctat 180
tgcgatgaaa ttaatgttat tatcgaaaag gataatagta ttacagtcaa agataacggt 240
cgtggaattc cggttggtat tcaagaaaaa atgggcagac ctgccgttga agttatctta 300
acggttcttc atgccggagg taaatttggc ggcggcggct ataaagtatc tggtggatta 360
cacggtgtag gtgcctcagt tgttaacgca ctttctacct ctttggaagt gcacgtacat 420
cgtgacggta aagttcatta tcaaaaatat gaacgaggtg taccggctgc tgacttaaaa 480
gtagttggag aaacagataa aacaggtact gttattcaat tccgtccaga cagtgaaatt 540
tttacagaaa cgcttgaata cgattttgac acgttagcta atcgtctgcg tgagttagct 600
ttcttaaatc gcggcattaa aattacgatt gaagacaaac gtgaagaaga taaaagacgt 660
gaatatcact atgaaggcgg aattaagtct tacgttgaac acttgaaccg ttcgaaagaa 720
gtgattcacg aagagcccat ctatattgaa ggtaatcgag acaacatttc tgtagaaatt 780
gctattcaat ataacgatag ctatacaagt aatttatatt cttttgcaaa caacattcac 840
acatatgaag gtggaacgca cgaagcagga tttaaaacag cgttaacgcg tgtaattaac 900
gactatgcac gtaaaaacag cgtatttaaa gacagtgacg ccaatctaac gggtgaagat 960
gttcgtgaag gaattacagc tatcatctct attaagcacc cagatccgca gttcgaagga 1020
caaacaaaaa caaagctggg aaatagtgaa gcaagaacaa ttactgactc tgtgtttgca 1080
gaacacttag aaacttactt gctagagaac cctattgtgg cgaaaaaggt aattgaaaaa 1140
ggtttaatgg ctgcaagagc aagaatggcg gctaaaaaag ctcgtgagct tacacgccgt 1200
aaaagcgcgc ttgaaatttc aaacttaccg ggtaaattag cagattgttc atcaaaagat 1260
ccttctatta gcgaacttta tgtagtagag ggtgactctg caggaggttc agctaagcag 1320
ggaagaagcc gtcatttcca agctattttg cctttacgtg gtaaaattat caacgtagag 1380
aaagcgcgtt tagataaaat tttatctaat aacgaaattc gtacaatcat taccgctcta 1440
ggaacgggta ttggtgacga ttttgatatt tcgaaagccc gctaccataa aattgtgatt 1500
atgacagatg cagacgtaga cggtgcgcat attcgtacgc ttcttctaac gttcttctat 1560
cgctatatga gacagattat tgagcacgga tatgtttaca ttgcccagcc gcctctttac 1620
aaagttacac agggtaaaaa agtggagtac gcgtacaacg atcgtcaatt agaagaggta 1680
ttagcttctt tccctgaagg cgcaaaacca aaccttcagc gttacaaagg tttaggagag 1740
atgaatcctg aacaattatg ggaaacaaca atggatccag agttccgtac ccttcttcag 1800
gtgaacttgc aagatgcaat tgaagctgat gagacatttg aaattttaat gggcgacaaa 1860
gtagaaccac gccgtaattt cattgaagaa aatgctcagt acgtaaaaaa tcttgatatt 1920
taa 1923
<210> 17
<211> 3564
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33296
<400> 17
ttgacaggtc aactagttca gtatggacgc caccgccaac gcagaagtta tgctcgtatt 60
agtgaagttt tagagttacc gaacttaatc gagattcaaa cggcttcata ccaatggttt 120
ttagatgaag gtttacgaga aatgttccaa gatatttctc caattgatga ttttacaggt 180
aacttatcac tagaatttat tgattacagc ttaggtgagc caaagtattc agtaggagag 240
tccaaagaac gtgatgtaac ttatgcagca cctcttcgtg ttaaggtgcg gttaattaat 300
aaagaaacag gtgaagtaaa agaccaagat gtgttcatgg gagatttccc attgatgacc 360
gaaacaggta cctttgtaat taatggtgct gagcgtgtta tcgtatcaca gttggttcgt 420
tcaccaagtg tttactatag tggaaagctt gataaaaacg gtaaaaaagg atatacagca 480
actgttatcc ctaaccgtgg tgcatggcta gaatatgaaa ctgatgcaaa agatgtagta 540
tacgtgcgta ttgatcgtac tcgtaaactg ccagtaacag tgctgttacg cgcgctaggt 600
ttcggttctg accaagagat tatcgattta atcggtgata atgaatacat ccgtaatacg 660
cttgaaaaag ataatacgga aacaacggaa aaagcgctat tagaaatcta tgagcgttta 720
cgtccaggtg aaccaccgac agttgagaat gcgaagtctc tattagtatc tcgcttcttt 780
gatccaaaac gatatgactt agcgaacgta ggtcgctata aaattaataa aaagcttcat 840
atcaaacatc gcttatttaa tcaaaagcta gctgaaacat tagttgatcc tgaaacaggt 900
gaaattattg cagaaaaagg cgcaatgatt gaccgccgtc tgctagatcg cttgattccg 960
atgcttgaag gtggagtaaa cttcaaaact tatagtccgg ttggtggagt agtagaagac 1020
gatgttacat tacaatctat taagatttat gcgccaaatg atccagaagg tgaaaaaatc 1080
atcactgtat caggtaacgc gtatgtaaca gaagaagtta aaaatatcac gcctgctgat 1140
attttagcat caatcagtta cttctttaac ttgcttcatc aagtaggaga cacagatgat 1200
atcgaccact taggtaaccg tcgtctgcgt tctgtaggtg aattgttaca aaaccaattc 1260
cgtatcggtt tatctcgtat ggaacgtgtt gttcgtgaaa gaatgtcaat tcaagacacg 1320
aatacaatca cacctcaaca attaattaat attcgcccag ttattgcggc gattaaagag 1380
ttctttggaa gttctcaatt atcacagttc atggatcaaa cgaatccatt aggcgaattg 1440
acgcacaaac gtcgtctttc agctctagga cctggtggtt taacgcgtga gcgcgctggt 1500
tttgaagtgc gtgacgttca ctactcccac tatggacgta tgtgtccgat tgaaacgcca 1560
gagggtccga atatcgggtt aatcaactca ctatcttctt atgcaaaagt aaacaaattc 1620
ggtttcatcg aaacacctta ccgtcgtatc gatcctgaaa caggtaaagt gacagagcga 1680
attgactact taacagctga tgaagaagat aactatgttg tagcccaagc gaacgctcgt 1740
ctaggtgatg atggttcatt cttagatgaa aatgtcgttg cacgtttccg tggagaaaac 1800
acggttatcc gtcgcgatcg tttagattat atggatgtat caccaaaaca agttgtatct 1860
gccgctacag catgtattcc attcttagag aacgatgact ctaaccgtgc attaatgggt 1920
gcgaacatgc aacgtcaagc agtaccttta ttaaatcctg aagcaccaat cgtaggtaca 1980
ggtatggaat acgtatctgg taaagactct ggtgcagccg tgatttgtaa atatcctggt 2040
gttgtagagc gcgtagaagc aaaacaaatt tttgttcgcc gctatgaaga agtagacgga 2100
caaaaagtta aaggtaactt agatcaatac aaattattaa aatttgttcg ttctaaccaa 2160
ggtacttgtt acaaccagcg tccaattgtt tcagttggcg acgaagtagt aaaaggtgag 2220
atcttagccg acggtccttc aatggaaaaa ggtgagcttg ctttaggacg aaacgtaatg 2280
gttggtttca tgacatggga tggttacaac tatgaggatg ccatcatcat gagtgaacgc 2340
cttgtgaaag acgatgtata tacgtctgtt catattgaag aatatgaatc tgagtctcgt 2400
gatacgaagc ttggacctga agaaattacg cgtgacattc caaacgtagg tgaagatgcg 2460
cttcgtaact tagatgagcg tggaatcatc cgcattggtg cagaagtaaa agacggagat 2520
cttttagttg gtaaagtaac gccaaaaggt gtaacagaac taacagctga agaacgtctt 2580
ctacacgcta ttttcggtga aaaagcgcgt gaagttcgtg atacttctct tcgtgtaccg 2640
cacggcggcg gtggaatcat tcttgatgtt aaagtcttca accgtgaaga tggcgacgaa 2700
ttaccaccag gtgtaaacca attagtccgt gtatatattg ttcagaagcg taaaatttct 2760
gaaggtgaca aaatggccgg tcgtcacggt aacaaaggtg taatttcacg tattttacct 2820
gaagaagata tgccttatct accagacggt acgccaattg acatcatgtt aaacccatta 2880
ggggtaccat ctcgtatgaa catcggtcag gtgctagagc ttcatttagg tatggctgct 2940
cgtaagcttg gcattcacgt tgcgtctcca gtatttgatg gtgcgcgtga ggaagatgtt 3000
tgggcaacaa tcgaagaagc tggcatgtct cgtgatgcta aaacagttct atatgatggt 3060
cgaacaggtg aaccattcga taaccgtgta tcagtaggaa tcatgtacat gatcaaactt 3120
gctcacatgg tagacgataa acttcacgct cgttctactg gaccatactc acttgttaca 3180
caacaaccac ttggtggtaa agcgcagttc ggtggacagc gttttggtga gatggaggta 3240
tgggcacttg aagcatacgg tgctgcttac acattacaag agatcttaac agtgaaatca 3300
gatgacgtag taggtcgtgt gaaaacatac gaagcaattg ttaaaggtga aaacattcca 3360
gaacctggca tacctgaatc gttcaaagta ttaattaaag aactgcaaag tttaggtatg 3420
gatgtgaaga tgctttctgc tgacgagcaa gaaattgata tcatggactc agaagaggac 3480
catgagcaac caacagaatc aattattgct gataacgaag aaagcctttc tgaaggacaa 3540
aaagatcctg tcacaaaaga gtaa 3564
<210> 18
<211> 1632
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33296
<400> 18
atggcaaaag acattaaatt tagcgaagaa gcacgtcgcg caatgctacg tggtgtagat 60
acattagcaa atgctgtaaa agtaacgctt ggaccaaaag gtcgtaacgt tgtattagaa 120
aagaaattcg gttcaccgct tattacaaat gacggtgtaa caattgcaaa agaaatcgaa 180
ttagaagacg catttgaaaa tatgggtgct aaattagtag ccgaagttgc tagcaaaaca 240
aacgacgttg ctggtgacgg tacaactact gcaacagttt tagcgcaagc aatgatcaga 300
gaaggtctta aaaacgtaac ggctggtgct aacccaatgg gtatccgtaa aggtatggaa 360
aaagcagtag ctgtagcggt tgaagaacta aaagcaatct ctaaaccaat tcaaggtaaa 420
gattcaattg ctcaagtagc ggctatctca gcagctgacg aagaagtagg tcaattaatt 480
gctgaagcaa tggagcgcgt tggtaacgac ggcgttatca cacttgaaga atcaaaaggt 540
ttcacaactg aattagaagt ggtagaaggt atgcagtttg accgtgggta tgcatctcct 600
tacatggtaa ctgattcaga taaaatggaa gctgtattag atgatccata catcttaatc 660
acagacaaaa aaatcggtaa cattcaagaa atcttaccgg tattagagca agttgttcaa 720
caaggcaagc ctctattaat catcgctgaa gacgtagaag gcgaagcttt agcaacatta 780
gttgtgaaca aacttcgtgg tacattcaca gctgtagctg ttaaagctcc tggtttcggt 840
gatcgtcgta aagcaatgct acaagacgtt gcgatcttaa caggcggaga agtaatcact 900
gaagagcttg gtcttgactt aaaaacagca agcatcgatc aattaggtcg cgcttctaaa 960
attgtcgtaa caaaagaaaa tacaacagtt gtaaacggtg caggaaacgc agaagacatc 1020
ctagcacgcg taaaccaaat caaagctcag cttgaagaaa caacttcaga gtttgaccgt 1080
gaaaaattac aagagcgctt agcaaaactt gctggtggcg tagctgtaat taaagttggt 1140
gcggcaactg aaactgagtt aaaagaacgt aaattacgta ttgaagatgc attaaactct 1200
acgcgtgctg cggttgaaga aggtatcgta gctggtggtg gtactgcatt agtaaatatc 1260
tataataaag tagcaagcat cgaagctgat ggtgacactg ctacaggtat caacatcgta 1320
ttacgtgcga ttgaagagcc tgtacgccaa attgctcaca acgctggttt agaaggatca 1380
gtaatcgttg agcgtctaaa aggcgaagct gttggaactg gatttaacgc tgcaactggc 1440
gagtgggtaa atatgctaga cactggtatc gttgacccaa caaaagtaac gcgttcagct 1500
cttcaaaatg cttcttctgt agcggctatg ttcttaacaa ctgaagcagt tgttgctgac 1560
aagccagaag aaggcggagc acctgcaatg cctgacatgg gcggcatggg tggaatgggc 1620
ggcatgatgt aa 1632
<210> 19
<211> 3150
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33296
<400> 19
atgacaatta aagaaatgcc tcagccaaaa acgtttggag agcttaaaaa tttaccgtta 60
ttaaacacag ataaaccggt tcaagctttg atgaaaattg cggatgaatt aggagaaatc 120
tttaaattcg aggcgcctgg ccgtgtaacg cgttacttat caagtcagcg tctaattaaa 180
gaagcatgcg atgaatcacg ttttgataaa aacttaagtc aagcgcttaa atttgtacgt 240
gattttgcag gagacgggtt atttacaagc tggacgcacg aaaaaaattg gaaaaaagcg 300
cataatatct tacttccaag cttcagtcag caggcgatga aaggctatca tgcgatgatg 360
gtcgatatcg ccgtgcagct tattcaaaag tgggagcgtc taaatgcaga tgagcatatt 420
gaagtaccgg aagatatgac gcgtttaacg cttgatacaa tcggtctttg cggctttaac 480
taccgcttta acagctttta ccgagatcag ccgcatccgt ttattacgag tatggtccgt 540
gcactggatg aatcgatgaa caagctgcag cgagcaaatc cagatgaccc agcttatgat 600
gaaaacaagc gccagtttca agaagatatc aaggtaatga acaacctagt agataaaatt 660
attgcagatc gcaaagcaag cggtgaacaa agcgatgatt tgttaacaca tatgctaaac 720
ggaaaagatc cagaaacagg tgagccgctt gatgacgaga acattcgcta tcaaattatt 780
acattcttaa ttgcgggaca cgaaacaaca agcggtcttt tatcatttgc gctgtatttc 840
ttagtgaaaa atccacatgt attacaaaaa gcagcagaag aagcagcacg agttctagta 900
gatcctgttc caagctacaa acaagtcaaa cagcttaaat atgtgggcat ggtcttaaac 960
gaagcgctgc gcttatggcc aacggctcct gcgttttccc tatatgcaaa agaagatacg 1020
gtgcttggag gagaatatcc tttagaaaaa ggcgacgaac taatggttct gattcctcag 1080
cttcaccgtg ataaaacaat ttggggagac gatgtggaag agttccgtcc agagcgtttt 1140
gaaaatccaa gtgcgattcc gcagcatgcg tttaaaccgt ttggaaacgg gcagcgtgcg 1200
tgtatcggtc agcagttcgc tcttcatgaa gcaacgctgg tacttggtat gatgctaaaa 1260
cactttgact ttgaagatca tacaaactac gagctggata ttaaagaaac tttaacgtta 1320
aaacctgaag gctttgtggt aaaagcaaaa tcgaaaaaaa ttccgcttgg cggtattcct 1380
tcacctagca ctgaacagtc tgctaaaaaa gtacgcaaaa aggcagaaaa cgctcataat 1440
acgccgctgc ttgtgctata cggttcaaat atgggaacag ctgaaggaac ggcgcgtgat 1500
ttagcagata ttgcaatgag caaaggattc gcaccacagg tcgctaccct tgattcgcac 1560
accggaaatc ttccccgcga aggagctgta ttaattgtaa cggcgtctta taacggacac 1620
ccgcctgata acgcaaagca atttgtcgac tggttagacc aagcgtctgc tgatgaagta 1680
aaaggcgttc gctattccgt atttggatgc ggtgataaaa actgggcgac aacgtatcaa 1740
aaagtgcctg cttttatcga tgaaacgctt gccgctaaag gggcagaaaa catcgctgac 1800
cgcggtgaag cagatgcaag cgacgacttt gaaggcacat atgaagaatg gcgtgaacat 1860
atgtggagtg atgtagcagc ctactttaac ctcgacattg aaaacagtga agataataaa 1920
tctacacttt cacttcaatt cgtcgacagc gccgcggata tgccgcttgc gaaaatgcac 1980
ggtgcgtttt cagcgaacgt cgtagcaagc aaagaacttc aacagccagg cagtgcacga 2040
agcacgcgtc atcttgaaat tgaacttcca aaagaagctt cttatcaaga aggagatcat 2100
ttaggcgtta ttcctcgcaa ctatgaagga atagtaaacc gtgtaacagc aaggttcggc 2160
ctagatgcat cacagcaaat ccatctggaa gcagaagaag aaaaattagc tcatttgcca 2220
ctcgctaaaa cagtatcagt agaagagctt ctgcaatacg tggagcttca agatcctgtt 2280
acgcgcacgc agcttcgcgc aatggctgct aaaacggtct gcccgccgca taaagtagag 2340
cttgaagcct tgcttgaaaa gcaagcctac aaagaacaag tgctggcaaa acgtttaaca 2400
atgcttgaac tgcttgaaaa atacccggcg tgtgaaatgg agttcagcga atttatcgcc 2460
cttctgccaa gcatacgccc gcgctattac tcaatttctt catcacctcg tgtcgatgaa 2520
aaacaagcaa gcatcacggt cagcgttgtc tcaggagaag cgtggagcgg atatggagaa 2580
tataaaggaa ttgcgtcgaa ctatcttgcc gagctgcaag aaggagatac gattacgtgc 2640
tttatttcca caccgcagtc aggatttacg ctgccaaaag accctgaaac gccgcttatc 2700
atggtcggac cgggaacagg cgtcgcgccg tttagaggtt tcgtgcaggc tcgcaaacag 2760
ctaaaagaac aaggacagtc gcttggagaa gcgcatttat acttcggctg ccgttcacct 2820
catgaagact atctgtacca agaagagctt gaaaacgccc aaaatgaagg catcattacg 2880
cttcataccg ctttttctcg cgtgccaaat cagccgaaaa catacgttca gcacgtgatg 2940
gaacaagacg gcaagaaatt gattgaactt cttgatcaag gagcgcactt ctatatttgc 3000
ggagacggag gccaaatggc acctgacgtt gaagcaacgc ttatgaaaag ctatgctgac 3060
gttcaccaag tgagtgaagc agacgctcgc ttatggctgc agcagctaga agaaaaaggc 3120
cgatacgcaa aagacgtgtg ggctgggtaa 3150
<210> 20
<211> 1812
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33296
<400> 20
gtgttgcaac ttaaggtagt aaacagccct tttaatcaag aacaagcaga tttgcttaat 60
cgccttctgc cgacattaac agaagcacaa aaaatgtggt tgagcggtta tttaacagca 120
tctcaatcta cgtctgccga aggaacgcca gacgtttcta cagcagcgcc tgctcaaacg 180
aagcagacag tttcaaaaga cgtaacgatt ctttacggat cacaaacagg aaatgctcaa 240
ggacttgctg aaaatacagg caaaacgctt gaagcaaaag gctttaatgt aactgtatct 300
tctatgaatg atttcaaacc aaataattta aagaaacttg aaaatttatt aattgtcgta 360
agtacacatg gagaaggaga gccgcctgat aatgcgctat ctttccatga atttcttcac 420
ggccgtcgag cgccaaaact tgagaacttc cgtttctctg ttttatcgct tggagacagc 480
tcatacgaat ttttctgtca aacaggaaaa gaatttgatg tgcgcttagc agaacttggg 540
ggtgaaagac tgtatccgcg cgttgactgt gatttagatt ttgaagagcc cgcaaataaa 600
tggcttaaag gtgttattga cggattaagc gaagcgaaag gacacagcgc ttcggcagct 660
gttccagcgg aagctcctgc aggaacttca ccatattcaa gaacaaatcc ttttaaagca 720
gaagtgcttg agaacttaaa cttaaacggc cgcggatcaa ataaagaaac gcgacactta 780
gaactatctc tagaaggttc aggtttgacg tatgaaccag gagacagttt aggtatttat 840
cctgaaaatg acccggagct tgttgatctt cttcttaacg aattcaagtg ggatgcaagt 900
gaaagtgtaa cggttaataa agaaggagaa acgcgtcctc ttagagaagc gctaatctct 960
aattttgaaa ttaccgtttt aacaaagcct cttttaaagc aagcagctga gcttactgga 1020
aacgataagt taaaagcgct tgtagaaaat cgcgaggaat taaaagcata cacacaaggc 1080
cgtgatgtaa ttgacttagt tcgtgacttc ggtccatgga acgtatcggc acaagagttt 1140
gtagccattc tacgtaaaat gccagcgcgt ctttactcga ttgcaagcag cttatcagca 1200
aaccctgatg aagttcatct aacaatcgga gcggtacgct acgaagcgca tggacgggag 1260
cgcaaaggtg tttgttcagt cctttgttca gaacgtttgc agccaggcga tacgattcct 1320
gtataccttc aaagcaataa aaactttaag cttcctcaag atcaagaaac gccaattatt 1380
atggtaggac ctggtacagg tgtggctccg ttccgctcat ttatgcaaga gcgtgaagaa 1440
acaggggcaa aaggcaagtc atggatgttc tttggagatc agcacttcgt aacagacttc 1500
ctttaccaaa cagaatggca aaagtggtta aaagatggcg tactgacaaa aatggacgtg 1560
gcgttttcac gcgatacaga agaaaaagta tacgtacaaa accgtatgct cgaacatagt 1620
aaagaattat tccaatggtt agaagaaggc gcatcttttt atgtgtgcgg agataaaaca 1680
aatatggcac gcgacgtgca caacacgcta gttgaaatta tcgaaacaga aggcaagatg 1740
agccgtgaac aggcggaagg ttaccttgct gaaatgaaga aacaaaaacg ttatcagcgt 1800
gatgtatact ga 1812
<210> 21
<211> 1278
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33296
<400> 21
ttgccggtta tacagggccc ctcgtcgtat aagcttaccg gtcacctgca aaagtttaga 60
gagaacccac tgggtttttt ggaaaacttg actcagtatg gagaaattgc aacatttaga 120
gttgcgcaca aacgctttta tgtaacgaga gaccctcaac ttattaaaga tgtagtcatt 180
acaaacagca aggcgtttca aaaaattaaa ctaacgcata tgtttaaaac gctgctaggc 240
gaagaaatgt tatggacaga tgaagcatta tatatgagcc ccattcaacc ctcacaacta 300
aaacaacatc taacttataa taaggaagca atcgcaaaaa taattgaaaa gcatacggaa 360
acgtgggaag aagggcagct tcgaacgata gtaaaagaca taagacagat agtcgtagct 420
gtactccttc aacttgtatt tggcatttcg attgaggaga aagataaaat tcactatgtt 480
caagcgctta tgagaaaaaa agaaaagtta ggcaaaattt atattcgtct acctttgcac 540
cagcttgact cagatgagca gctagaacaa ctcctttttg aacgcgttca aatgcgtgtt 600
caaaacaaaa ccgaaggaaa tgatttactg cagtatatat tgaattctta cggagaagac 660
agcgatgaaa gagaaatata tgaacagcta aattctatat tcctttcgat gtatgaaatg 720
attacgcacg tatgcagctg gtctattcat ttactgtctc aaaatactag ggagcacctt 780
cagctgtata aagaaattca agcctatgct agcggcgaat cactttcaac caaaaatctg 840
acctatatgc gcaaaataat tgctgaaagc atgaggttgt atccgcctct ttggctattt 900
gggcgtcaag cacgtgaaga tatacaaata gatggctaca gtatcaaaaa aggggagatt 960
atgttaatca gcccttatat gatgcatcgc catgaagatt attttttaga gccaagtgaa 1020
tttttacctg accgttttga aaaaggaggg tccattgatg tcccaagcta tatgtatatg 1080
ccgcttggga ttgagcatca agcggaaagg ggcattaact atatcactga aatagtaacc 1140
atttttctgt cagaaatgac aaagcgcttt ttatttcagc tgactgagcc cgaatctatt 1200
gctccgatgg cgggcgtaat gctgaatatg aaagaagaac tgaaggtgaa tgttcacaaa 1260
gttcacgcgc agtcttga 1278
<210> 22
<211> 1215
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33296
<400> 22
atgaaaaccg aaagagaaaa cggaattgtc cgtcaagtga atacaattca aacaaaagaa 60
gagcgcttta atcctttttc atggtatgaa gagatgagaa acaccgcgcc tgtgcaatgg 120
gatgaagaaa ggcaggtatg ggatgttttt cactatgacg gggttaaaga agtgctagaa 180
caaaagaata ttttttcttc tgatcgaaga cctccacaaa accaaagaca aactgctcta 240
ggaacgagtc taattaatat tgatccgcct aagcatgctg aaatgagagc acttgttaat 300
aaagctttta cgcctaaagc aatgaaagca tgggagccta aaattgcgcg tattacagct 360
gaattattac aagaagttga gcatcttgaa gatattgata tagtcgagca tctgtcctat 420
ccgcttccgg ttatggtaat tgccgatatt ttaggcgtgc cgatagaaga tcagcgtcag 480
tttaaagatt ggtccgatat tatcgtagcc ggtccttcta ataatgaacg tgaaacgctc 540
gaaaaattgc agcaagagaa aatgaaagca aatgatgagc tagaaactta tttttatcga 600
atcattgaag aaaaacgcac gcgtccagga gatgatatta tctccgtgct tcttcaggca 660
aaagaagaag ggaaacagct aacggatgaa gaaatcgtcg gtttttctat tttgctgctg 720
attgctggaa acgaaacaac cacaaattta atttcaaata cgatttattg tttaatggaa 780
gataaaactt cttttgaacg actcaaacga gagaaagaac ttttgccttc tgcgattgaa 840
gaagttcttc gctatcgttc acccgttcag gctcttcacc gaatcgtaaa agaagatgtg 900
actcttgcag gcaagaaact aaaagcgggc gaacacgtgg ttccatggat gggatctgcg 960
catcgagacg cggagtattt tgaagacccg gatgtattta aaatcgatcg aaagccaaat 1020
gtgcacatgg catttggaag aggcattcat ttttgcttag gagcacccct tgctcgaata 1080
gaagcaaaaa ttatgctggc cgagctgatt gaccgctatc cgcagatgga ctggagcccg 1140
tcatttgggt taaacccgat tgaaagcacg tttgtgtacg ggttaaaaga attattgatt 1200
cgtaaaaacg tataa 1215
<210> 23
<211> 1212
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33296
<400> 23
atgaattcaa aagcagtaaa aagagaaaat cgctatgcta acttgattcc tatgcaagaa 60
atcaaatcag ttgaacagca gctttaccca tttgatattt ataacagtct gcgtcaaaag 120
gcacctattc gttacgatga aagccgaaac tgctgggatg tatttgatta tgaaacagta 180
aagtatattt taaaaaaccc gtcgcttttt tcatccaaac gagcaatgga agaacgccaa 240
gaaagtattt taatgatgga ccctccaaaa catacaaagt tacgaaactt agtaaacaaa 300
gcttttacgc caagagctat tcagcactta gagagtcata ttgaagagat tgcagattat 360
ttattagatg aagtaagcag ggaaaaaaag tttgatatag tagaagactt tgctggacca 420
ctgccgatta ttgtaatagc agaattgcta ggagtaccaa tccaagaccg agaactgttt 480
aaaaaatact ccgatgactt agtgagcggt gctgaaaata attcagacga agcttttgcg 540
aaaatgatgc aaaagcgtaa tgaaggcgtg atttttttac aagggtattt taaagaaatt 600
atcgcggagc gtcagcaaaa taaacaagaa gatctcattt cgcttctttt agaagcagaa 660
atcgacggcg agcatttaac agaagaggaa gtgctggggt tttgtatttt attgctagta 720
gcaggtaatg agacaacgac aaacttaatt acgaacggag tccggtatat gacagaagac 780
gtagacgtgc agaatgaagt tcgtcaagac atatcgctag ttccgaacct ggtagaagaa 840
acgcttcgct attatcctcc tattcaagcg attggacgca tagctgctga agatgtagaa 900
cttggagagt gtaagattaa aaaagggcag caggtaatta gctgggcagc aagtgcaaat 960
agagattcag ctaagtttga acggcctgac acttttgtcg tgcatcgtaa aacaaatcct 1020
cacgtgagct ttggattcgg catccatttt tgcttaggag caccgctagc aagaatggaa 1080
gggaaaattg cttttactaa gttactagag aaaggcgagt ttttaaaagt tgagaatcag 1140
tctttaaagc caataggcag cccttttgtt tttggcgtaa aaaaatatga gattactttt 1200
aataaagctt aa 1212
<210> 24
<211> 1233
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33296
<400> 24
atgaacaaag aagtcattcc cgttacagaa attccaaaat tccaatcacg tgcagaagag 60
tttttcccta tccaatggta taaagaaatg ctgaataacc accctgttta ttttcatgaa 120
gaaacaaaca catggaatgt attccaatac gaccatgtta aacaagtctt gagcaactac 180
gaattctttt caagcgatgg gcaaagaacc actatttttg ttggggataa tagtaagaaa 240
aaaagcacat ctccaattac caatcttaca aacttagatc ctcctgacca ccggaaagcg 300
cgatctttgc ttgctgcagc ctttactcct cgcagtttaa agaactggga accgcgaatc 360
aaacaaattg cagcagacct tgtagaagcc atacaaaaga attcaactat taatattgtt 420
gacgatttat cttctccttt tcctagttta gttatagcag atttattcgg tgtaccggta 480
aaagaccaat atcagttcaa aaaatgggta gatattctgt ttcagcctta tgaccaagaa 540
agattagaag agattgaaca agaaaagcag cgtgcaggag ctgaatattt tcaatacctc 600
tacccgattg tcattgaaaa gcgctccaac ctttctgatg atattatctc agacttaatt 660
caagccgaag ttgatggtga aacatttaca gatgaagaaa ttgtacacgc taccatgctg 720
cttttaggtg caggagttga aaccacaagt cacgccattg caaatatgtt ttattctttt 780
ttatatgacg acgagtcgtt atacagcgag ctgagaaaca atagagaatt agctccaaaa 840
gcagtagaag aaatgcttcg atatcggttt cacatctcca gacgagatcg gacggttaaa 900
caagataacg atttattagg cgttaagcta aaaaaaggag atgtcgttat tgcgtggatg 960
agtgcatgta atatggatga gaatatgttt gaaaaccctt tttctgtaga cattcaccgc 1020
caaactaata aaaaacactt aacgtttgga aacggacctc atttttgttt gggcgcccct 1080
cttgcccgac ttgaaatgaa aatcatatta gaagcatttt tagaggcctt ttctcatatc 1140
gaaccatttg aagattttga attagaatcg catttaactt cttcagctac ggggcaatct 1200
ttaacgtatt tgccgatgaa ggtgtacaga taa 1233
<210> 25
<211> 1549
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33299
<400> 25
tcggagagtt tgatcctggc tcaggatgaa cgctggcggc gtgcctaata catgcaagtc 60
gagcgaactg attagaagct tgcttctatg acgttagcgg cggacgggtg agtaacacgt 120
gggcaacctg cctgtaagac tgggataact tcgggaaacc gaagctaata ccggatagga 180
tcttctcctt catgggagat gattgaaaga tggtttcggc tatcacttac agatgggccc 240
gcggtgcatt agctagttgg tgaggtaacg gctcaccaag gcaacgatgc atagccgacc 300
tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac gggaggcagc 360
agtagggaat cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa 420
ggctttcggg tcgtaaaact ctgttgttag ggaagaacaa gtacgagagt aactgctcgt 480
accttgacgg tacctaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 540
cgtaggtggc aagcgttatc cggaattatt gggcgtaaag cgcgcgcagg cggtttctta 600
agtctgatgt gaaagcccac ggctcaaccg tggagggtca ttggaaactg gggaacttga 660
gtgcagaaga gaaaagcgga attccacgtg tagcggtgaa atgcgtagag atgtggagga 720
acaccagtgg cgaaggcggc tttttggtct gtaactgacg ctgaggcgcg aaagcgtggg 780
gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta 840
gagggtttcc gccctttagt gctgcagcta acgcattaag cactccgcct ggggagtacg 900
gtcgcaagac tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 960
tttaattcga agcaacgcga agaaccttac caggtcttga catcctctga caactctaga 1020
gatagagcgt tccccttcgg gggacagagt gacaggtggt gcatggttgt cgtcagctcg 1080
tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt agttgccagc 1140
atttagttgg gcactctaag gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg 1200
tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaaag 1260
ggctgcaaga ccgcgaggtc aagccaatcc cataaaacca ttctcagttc ggattgtagg 1320
ctgcaactcg cctacatgaa gctggaatcg ctagtaatcg cggatcagca tgccgcggtg 1380
aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg taacacccga 1440
agtcggtgga gtaaccgtaa ggagctagcc gcctaaggtg ggacagatga ttggggtgaa 1500
gtcgtaacaa ggtagccgta tcggaaggtg cggctggatc acctccttt 1549
<210> 26
<211> 1549
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33299
<400> 26
tcggagagtt tgatcctggc tcaggatgaa cgctggcggc gtgcctaata catgcaagtc 60
gagcgaactg attagaagct tgcttctatg acgttagcgg cggacgggtg agtaacacgt 120
gggcaacctg cctgtaagac tgggataact tcgggaaacc gaagctaata ccggatagga 180
tcttctcctt catgggagat gattgaaaga tggtttcggc tatcacttac agatgggccc 240
gcggtgcatt agctagttgg tgaggtaacg gctcaccaag gcaacgatgc atagccgacc 300
tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac gggaggcagc 360
agtagggaat cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa 420
ggctttcggg tcgtaaaact ctgttgttag ggaagaacaa gtacgagagt aactgctcgt 480
accttgacgg tacctaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 540
cgtaggtggc aagcgttatc cggaattatt gggcgtaaag cgcgcgcagg cggtttctta 600
agtctgatgt gaaagcccac ggctcaaccg tggagggtca ttggaaactg gggaacttga 660
gtgcagaaga gaaaagcgga attccacgtg tagcggtgaa atgcgtagag atgtggagga 720
acaccagtgg cgaaggcggc tttttggtct gtaactgacg ctgaggcgcg aaagcgtggg 780
gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta 840
gagggtttcc gccctttagt gctgcagcta acgcattaag cactccgcct ggggagtacg 900
gtcgcaagac tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 960
tttaattcga agcaacgcga agaaccttac caggtcttga catcctctga caactctaga 1020
gatagagcgt tccccttcgg gggacagagt gacaggtggt gcatggttgt cgtcagctcg 1080
tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt agttgccagc 1140
atttagttgg gcactctaag gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg 1200
tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaaag 1260
ggctgcaaga ccgcgaggtc aagccaatcc cataaaacca ttctcagttc ggattgtagg 1320
ctgcaactcg cctacatgaa gctggaatcg ctagtaatcg cggatcagca tgccgcggtg 1380
aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg taacacccga 1440
agtcggtgga gtaaccgtaa ggagctagcc gcctaaggtg ggacagatga ttggggtgaa 1500
gtcgtaacaa ggtagccgta tcggaaggtg cggctggatc acctccttt 1549
<210> 27
<211> 1188
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33299
<400> 27
atgaagaatg gatggacaaa ctttgttatt ggtaccgtac gtattcgaat tgttggaaaa 60
ggaattgaac gttttttaaa taattgtgtg agacaacaga ttatgatttc gaatgtccat 120
aaagttgacg gtcagctagc gacagcaacg atattattga aagatgtaaa gaaaattcgt 180
atactaattc gcaacgcaga ctgtaaaatt tattttataa gaggtcgagg ttttcctttt 240
ttaacaaaac gagtgatcaa aaatagtggg tttgctttag gttttctttc tttttttatt 300
attcttggtt tgctttccaa catggtgtgg aaagtggaaa ttagcggtgc agaaccacaa 360
acagaacatc aaatgactaa gcagctagcc aaaataggtg taaaaagagg agagtttcaa 420
tttctgttag agagtcctga aaaaattcag cgctatttaa cggataatat gaacaatatt 480
acctgggtgg gagtagaagt gcgcggcact tcttatcact tccaagtggt tgaaaaaaat 540
gagcccaagc ctcagcaaaa aacaccttac caacatttaa ttgctaaaaa gaaagccatt 600
attacgaatt tgtttgtcga aaaaggacaa cctttagtca aggttaacga ttttgtcaat 660
gaaggcgagg tcttagtttc cggtattatt ggaaacgaaa aaaataaaaa agtagtagct 720
gctaaaggga aagtctatgg tgaaacatgg tataagtctg aggtggaagt acctttaaaa 780
actgattttc aggtattaac gggtaatgga tatacgaagc actatttaga ttttaaagct 840
tttaaaatgc ctctttgggc ttttaacaaa gagaaatacg gctcaaaggt gacagaaaaa 900
gtggaacatc cactctattt ttttaaatgg aaattacctc tgtcgtatga gaaagttgct 960
gtacgagaag aacagaactc gcagcgcgtc tattctaaac aagaggcggt ggaaaaagct 1020
ttggaaatcg gtcgaaaaaa attactatca actttaggtg aggatgctaa aataaaaggt 1080
gaaaaagttt tgcaccaaga acaggacaat ggtaaagtta gattatcaat acattaccaa 1140
gttatagaaa atattgcgaa cactcaacca attattcaag gagattga 1188
<210> 28
<211> 1923
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33299
<400> 28
ttgacgatgg aacaaaaaga agtacaagca tatgaagctg atcagataca agtattagaa 60
ggattagaag ctgttcgtaa acgtccgggg atgtatattg gatcgacgag cgctaagggt 120
ttacatcatc ttgtatggga aattgtagat aatagtattg atgaagcgct ggccggctat 180
tgcgatgaaa ttaatgttat tatcgaaaag gataatagta ttacagtcaa agataacggt 240
cgtggaattc cggttggtat tcaagaaaaa atgggcagac ctgctgttga agttatctta 300
acggttcttc atgccggagg taaatttggc ggcggcggct ataaagtatc tggtggatta 360
cacggtgtag gtgcctcagt tgttaacgca ctttctacct ctttggaagt acacgtacat 420
cgtgatggta aagttcatta tcaaaaatat gaacgaggtg taccagctgc tgacttgaaa 480
gtagtcggag aaacagataa aacaggtact gttattcaat tccatccaga cggtgaaatt 540
tttacagaaa cgcttgaata cgattttgat acgttagcta atcgtctgcg tgagttagct 600
ttcttaaatc gcggcattaa aattacgatt gaagacaaac gtgaagaaga taaaagacgt 660
gaatatcact atgaaggcgg aattaagtct tacgttgaac acttaaaccg ttcgaaagaa 720
gtgattcacg aagagccaat ctatatcgaa ggtaatcgag acaacatttc tgtagaaatt 780
gctattcaat ataacgatag ctatacaagt aatttatatt cttttgcaaa caacattcac 840
acatatgaag gtggaacgca cgaagcagga tttaaaacag cgttaacgcg tgtaattaac 900
gactatgcac gtaaaaacag cgtatttaaa gacagtgacg ccaatttaac gggtgaagat 960
gttcgtgaag gaattacagc tatcatctct attaagcacc cagatccgca gttcgaagga 1020
caaacgaaaa caaagctggg aaatagtgaa gcaagaacaa ttactgactc tgtgtttgca 1080
gaacacttag aaacttactt gctagagaac cctattgtgg cgaaaaaggt aattgaaaaa 1140
ggtttaatgg ctgcaagagc aagaatggca gctaaaaaag ctcgtgagct tacaagacgt 1200
aaaagcgcgc ttgaaatttc aaacttaccg ggtaaattag cagattgttc atcaaaagat 1260
ccttctatta gcgaactcta tgtagtagag ggtgactctg ccggcggttc agctaagcag 1320
ggaagaagcc gtcatttcca agctattttg cctttgcgcg gtaaaattat caacgtagag 1380
aaagcgcgtt tagataaaat tttatctaat aacgaaattc gtacaatcat taccgctcta 1440
ggaacgggta ttggtgacga ttttgatatt tcgaaagccc gctaccataa aattgtgatt 1500
atgacagatg cagacgtaga cggtgcgcat attcgtacgc ttcttctaac gttcttctat 1560
cgctatatga gacagattat tgagcacgga tatgtgtaca ttgcccagcc gcctctttac 1620
aaagtttcac aaggtaaaaa agtggagtac gcgtacaacg atcgtcaatt agaagaggta 1680
ttagcttctt tccctgaagg cgcaaaacca aaccttcagc gttacaaagg tttaggagag 1740
atgaatcctg aacaattatg ggaaacaaca atggatccag aattccgtac tcttcttcag 1800
gtgaacttgc aggatgcaat tgaagctgat gagacatttg aaattttaat gggcgacaaa 1860
gtagaaccac gccgtaattt cattgaagaa aatgctcagt acgtaaaaaa tctcgatatt 1920
taa 1923
<210> 29
<211> 3564
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33299
<400> 29
ttgacaggtc aactagttca gtatggacgc caccgccaac gcagaagtta tgctcgtatt 60
agtgaagttt tagagttacc gaacttaatc gagattcaaa cggcttcata ccaatggttt 120
ttagatgaag gtttacgaga aatgttccaa gatatttctc caattgatga ttttacaggt 180
aacttatcac tagaatttat tgattacagc ttaggtgagc caaagtattc agtaggagag 240
tctaaagaac gtgatgtaac ttatgcagca cctcttcgtg ttaaggtgcg gttaattaat 300
aaagaaacag gtgaagtaaa agaccaagat gtgttcatgg gagatttccc attgatgacc 360
gaaacaggta cctttgtaat taatggtgct gagcgtgtta ttgtatcaca gttggttcgt 420
tcaccaagtg tttactatag tggaaagctt gataaaaacg gtaaaaaagg atatacagca 480
actgttatcc ctaaccgtgg tgcatggcta gaatatgaaa ctgatgcaaa agatgtagta 540
tacgtgcgta ttgatcgtac tcgtaaactg ccagtaacag tgctgttacg cgcgctaggt 600
ttcggttctg accaagagat tatcgattta atcggtgata atgaatacat ccgtaatacg 660
cttgaaaaag ataacacgga aacaacggaa aaagcgctat tagaaatcta tgagcgttta 720
cgtccgggtg aaccaccgac agttgagaat gcgaaatctc tattagtatc tcgcttcttt 780
gatccaaaac gatatgactt agcgaacgta ggtcgctata aaattaataa aaagcttcat 840
atcaaacatc gcttatttaa tcaaaaacta gctgaaacat tagttgatcc tgaaacaggt 900
gaaattattg cagaaaaagg cgcaatgatt gaccgacgtc tgctagatcg cttgattccg 960
atgcttgaag gtggagtaaa cttcaaaact tatagtccag ttggtggagt agtagaagac 1020
gatgttacat tacaatctat taagatttat gcgccaaatg atccagaagg tgaaaaagtc 1080
atcactgtat caggtaacgc atatgtaaca gaagaagtta aaaatatcac acctgctgat 1140
attttagcat caatcagtta cttctttaac ttgcttcatc aagtaggaga cacagatgat 1200
atcgaccact taggtaaccg tcgtctgcgt tctgtaggtg aattgttaca aaaccaattc 1260
cgtatcggtt tatctcgtat ggaacgtgtt gttcgtgaaa gaatgtcaat tcaagacacg 1320
aatacaatca cacctcaaca attaattaat attcgtccag ttattgcggc gattaaagag 1380
ttctttggaa gttctcaatt atcacagttc atggatcaaa cgaatccatt aggcgaattg 1440
acgcacaaac gtcgtctttc agctctagga cctggtggtt taacgcgtga gcgcgctggt 1500
ttcgaagtgc gtgacgttca ctactcccac tatggccgta tgtgtccgat tgaaacgcca 1560
gaggggccga atatcgggtt aatcaactca ctttcttctt atgcaaaagt aaacaaattc 1620
ggtttcatcg aaacacctta ccgtcgtatc gatcctgaaa caggtaaagt gacagagcga 1680
attgactact taacagctga tgaagaagat aactatgttg tagcccaagc gaacgctcgt 1740
ctaggtgatg atggttcatt cttagatgaa aatgtcgttg cacgtttccg tggagaaaac 1800
acggttatcc gtcgcgatcg tttagactat atggatgtat caccaaaaca agttgtatct 1860
gccgctacgg catgtatccc attcttagag aacgatgact ctaaccgtgc attaatgggt 1920
gcgaacatgc aacgtcaagc agtaccttta ttaaatcctg aagcaccaat cgtaggtaca 1980
ggtatggaat atgtatctgg taaagactct ggtgcagccg tgatttgtaa ataccctggc 2040
gttgtagagc gcgtagaagc aaaacaaatt tttgttcgcc gctatgaaga agtagacgga 2100
caaaaggtta aaggcaactt agatcaatac aaattattaa aattcgttcg ttctaaccaa 2160
ggtacttgtt acaaccagcg tccaattgtt tcagttggcg acgaagtagt aaaaggtgag 2220
atcttagccg atggtccttc aatggaaaaa ggtgagcttg ctttaggacg aaacgtaatg 2280
gttggtttca tgacatggga tggttacaac tatgaggatg ccatcatcat gagtgaacgc 2340
cttgtgaaag acgatgtata tacgtctgtt catattgaag aatatgaatc tgagtctcgt 2400
gatacgaaac ttggacctga agaaattacg cgtgacattc caaacgtagg tgaagatgcg 2460
cttcgcaact tagatgagcg tggaatcatc cgcattggtg cagaagtaaa agatggagat 2520
cttttagttg gtaaagtaac gccaaaaggt gtaacagaac taacagctga agaacgtctt 2580
ctacacgcta ttttcggtga aaaagcgcgt gaagttcgtg atacttctct tcgtgtaccg 2640
cacggcggcg gtggaattat tcttgatgtt aaagtcttca accgtgaaga tggcgacgaa 2700
ttaccaccag gtgtaaacca attagtccgt gtatatattg ttcagaagcg taaaatttct 2760
gaaggtgaca aaatggccgg tcgtcacggt aacaaaggtg taatttcacg tattttaccg 2820
gaagaagata tgccttacct accagacggt acgccaattg acatcatgtt aaacccatta 2880
ggggtaccat ctcgtatgaa catcggtcag gtgctagagc ttcatttagg tatggctgct 2940
cgtaagcttg gcattcacgt tgcgtctcca gtatttgatg gtgcgcgtga ggaagatgtt 3000
tgggcaacaa tcgaagaagc tggcatgtct cgtgatgcta aaacagttct atatgatggt 3060
cgaacaggtg aaccattcga taaccgtgta tcagtaggaa tcatgtacat gatcaaactt 3120
gctcacatgg tagacgataa acttcacgct cgttctactg gaccatactc acttgttaca 3180
caacaaccac ttggtggtaa agcgcagttc ggtggacagc gttttggtga gatggaggta 3240
tgggcacttg aagcatacgg tgctgcttac acattacaag agatcttaac agtgaaatca 3300
gacgacgtag taggtcgtgt gaaaacatac gaagcaattg ttaaaggtga aaacattcca 3360
gaacctggca tacctgaatc gttcaaagta ttaattaaag aactacaaag tttaggtatg 3420
gatgtgaaga tgctttctgc tgacgagcaa gaaattgata tcatggactc agaagaggac 3480
catgagcaac caacagaatc aattattgca gataacgaag aaagcctttc tgaaggacaa 3540
aaagatcctg tcacaaaaga gtaa 3564
<210> 30
<211> 1632
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33299
<400> 30
atggcaaaag acattaaatt tagcgaagaa gcacgtcgcg caatgctacg tggtgtagat 60
acattagcaa atgctgtaaa agtaacgctt ggaccaaaag gtcgtaacgt tgtattagaa 120
aagaaattcg gttcaccgct tattacaaat gatggtgtaa caattgcaaa agaaatcgaa 180
ttagaagacg catttgaaaa catgggtgct aaattagtag ccgaggttgc tagcaaaaca 240
aacgacgttg ctggtgacgg tacaactact gcaacagttt tagcgcaagc aatgatcaga 300
gaaggtctta aaaacgtaac ggctggtgct aacccaatgg gtatccgtaa aggtatggaa 360
aaagcagtag ctgtagcggt tgaagaacta aaagcaatct ctaaaccaat tcaaggtaaa 420
gattcaattg ctcaagtagc ggctatctca gcagctgacg aagaagtagg tcaattaatt 480
gctgaagcaa tggagcgcgt tggtaacgac ggcgttatca cacttgaaga atcaaaaggt 540
ttcacaactg aattagaagt ggtagaaggt atgcagtttg accgtggata tgcatctcct 600
tatatggtaa ctgattcaga taaaatggaa gctgtattag atgatccata catcttaatc 660
acagacaaaa aaatcggtaa cattcaagaa atcttaccgg tattagagca agttgttcaa 720
caaggcaagc ctctattaat catcgctgaa gacgtagaag gcgaagcttt agcaacatta 780
gttgtgaaca aacttcgtgg tacatttaca gctgtagctg ttaaagctcc tggttttggt 840
gatcgtcgta aagcaatgct acaagacgtt gcgatcttaa caggcggaga agtaatcact 900
gaagagcttg gtcttgactt aaaaacagca ggcatcgatc aattaggtcg cgcttctaaa 960
attgttgtaa caaaagaaaa tacaacagtt gtaaacggtg caggaaacgc agaagatatc 1020
ctagcacgcg taaaccaaat caaagctcag cttgaagaaa caacttcaga gtttgaccgt 1080
gaaaaattac aagagcgctt agcaaaactt gctggtggcg tagctgtaat taaagtgggt 1140
gcggcaactg aaactgagtt aaaagaacgt aaattacgta ttgaagatgc attaaactct 1200
acgcgtgctg cggttgaaga aggtatcgta gctggtggtg gtactgcatt agtaaatatc 1260
tataataaag tagcaagcat cgaagctgac ggtgacactg ctacaggtat caacatcgta 1320
ttacgtgcga tcgaagagcc tgtacgtcaa atcgctcaca atgctggttt agaaggatca 1380
gtaatcgttg agcgtctaaa aggcgaagca gttggaactg gattcaatgc tgcaactggc 1440
gagtgggtaa acatgctaga cactggtatc gttgacccaa caaaagtaac gcgttcagct 1500
cttcaaaatg cttcttctgt agcggctatg ttcttaacaa ctgaagcagt tgttgctgac 1560
aagccagaag aaggcggagc acctgcaatg cctgacatgg gcggcatggg tggaatgggc 1620
ggcatgatgt aa 1632
<210> 31
<211> 3150
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33299
<400> 31
atgacaatta aagaaatgcc tcagccaaaa acgtttggag agcttaaaaa cttaccgtta 60
ttgaacacag acaaaccact tcaaacgctg atgaaaattg cggatgaatt aggggaaatc 120
tttaaatttg aagcgcctgg ccgtgtaacg cgctacttat caagtcagcg tctaattaaa 180
gaagcatgtg atgagtcccg attcgataaa aacttaagtc aagctcttaa atttgtgcgt 240
gattttgcag gagacggttt atttacaagc tggactcatg agaaaaactg gaaaaaagca 300
cacaacatct tacttccaag ctttagccag caggcaatga aaggctatca cgcaatgatg 360
gtcgatatcg ccgtgcagct tattcaaaaa tgggagcgtc taaatgcaga tgagcatatt 420
gaagtaccgg aagacatgac gcgcttaacg cttgacacaa tcggtctttg cggctttaat 480
taccgcttta acagctttta tcgcgatcag ccgcacccgt ttattacaag tatggtccgt 540
gcactggatg aatcgatgaa aaagctgcag cgtgcaaatc cggatgaccc agcttatgat 600
gaaaataagc gccagtttca agaagatgtt aaggtgatga acaacctagt agataaaatt 660
attgcagatc gcaaagcaag cggtgaacaa agtgatgatc tattaacgca tatgctaaac 720
ggaaaagatc cagaaacagg tgagccgctt gatgacgaga acattcgcta tcaaattatt 780
acgttcttaa ttgcgggaca cgaaacaaca agcggtctgt tatcatttgc gctgtatttc 840
ttagtgaaaa atccacatgt attacaaaaa gcagcagcag aagcagcacg agttctagta 900
gatcctgttc caacctacaa acaagtcaaa cagcttaaat atgtgggcat ggtcttaaat 960
gaagcgctgc gcttatggcc gacggctcct gcgttttctc tatatgcaaa agaggatacg 1020
gtgcttggag gagaatatcc gttagaaaaa ggggatgaac tgatggttct gattcctcag 1080
cttcaccgtg ataaaacgat ttggggagac gatgtagaag agttccgccc agagcgtttt 1140
gaaaacccaa gcgcgattcc gcagcatgcg tttaaaccat ttggaaacgg tcagcgcgcg 1200
tgtatcggtc agcagttcgc tcttcatgaa gcaacgcttg tacttggtat gatgctaaaa 1260
cactttgact ttgaagatca tacaaactac gagctagata ttaaagaaac gttaacgtta 1320
aaacctgagg gctttgtggt aaaagcaaaa tcgaaacaaa ttccgcttgg cggcattcct 1380
tcaccaagcc gagagcagtc agctaaaaaa gagcgcaaaa ccgtagaaaa cgctcataat 1440
acgccgctgc ttttgctata cggttcaaat atgggaacag ccgaaggaac ggcgcgggat 1500
ttagcggata ttgcgatgag caaaggattc gcaccgcaag tcgcaacgct tgattcccac 1560
gcaggaaatc ttccgcgtga aggagctgtg ttaattgtaa cagcttcgta taacggacat 1620
ccgcctgata acgcaaagca gtttgtcgag tggttagacc aagcgtctgc tgatgaagtc 1680
aaaggcgttc gctactccgt atttggatgc ggcgataaaa actgggctac tacgtatcaa 1740
aaagtgcctg cttttattga tgaaacgctt gcggctaaag gagcagaaaa catcgctgaa 1800
cgcggcgaag cagatgcaag cgacgacttt gaaggcacat acgaagaatg gcgtgaacac 1860
atgtggagtg acgtagcagc ctactttaac ctcgacattg aaaacagtga agataatcaa 1920
tctacgcttt cacttcaatt tgtcgacagc gctgcggaca tgccgcttgc gaaaatgcac 1980
ggtgcgtttt cagcaaacgt cgtagcgagc aaagaacttc aacagccagg cagtgcacga 2040
agcacgcgac accttgaaat tgaacttcca aaagaagctt cttatcaaga aggagatcat 2100
ttaggtatta ttcctcgcaa ctatgaagga atagtaaatc gtgtaacagc aaggttcgat 2160
gtagatgcat cacagcaaat ccgtctggaa gctgaagaag aaaaattagc gcatttgcca 2220
ctcggtaaaa cagtatcagt agaagagctt ctacaatatg tagagcttca agatcctgtt 2280
acgcgcacgc agcttcgcgc aatggctgct aaaacggtct gcccgccgca taaagtggag 2340
cttgaagcct tgcttgaaaa gcaagcgtac aaagaacaag tactggcaaa acgcttaaca 2400
atgcttgaac tgcttgaaaa atacccggcg tgtgaaatgg aattcagcga atttatcgct 2460
cttcttccaa gcatgcgtcc gcgctattac tcaatttctt catcaccttg cgtcgatgaa 2520
aaacaagcaa gcatcacggt cagcgttgtt tcaggagaag cgtggagcgg atatggagaa 2580
tacaaaggaa ttgcgtcgaa ctatcttgcc gagctgcaag aaggagatac gattacgtgc 2640
tttgtttcca caccgcagtc aggctttacg ctgccaaaag aatctgaaac acctattatc 2700
atggtcggac cgggaacagg cgtcgcgcca tttagaggct ttgtgcaggc tcgcaagcag 2760
ttaaaagaac aaggacagtt gcttggagaa gcgcatttat actttggctg ccgttcacct 2820
catgaagact atctgtatca agaagagctt gaaaacgcac aaaatgaagg tgtcattacg 2880
cttcataccg ctttttcacg cgtaccaaat cagccgaaaa catacgttca gcgcctgatg 2940
gaacaagacg gtatgaaatt gattgaactt cttgatcaag gagcgcattt ctacatttgc 3000
ggagacggaa gccaaatggc acctgacgtt gaagcaacgc ttattaaaag ctatgctgat 3060
gttcataaag taagtgaagc agacgctcgc ttatggctgc agcagctaga agaaaagggc 3120
cgatacgcaa aagacgtgtg ggctgggtaa 3150
<210> 32
<211> 1812
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33299
<400> 32
gtgttgcaac ttaaggtagt aaacagccct tttaatcaag aacaagcaga tttgcttaat 60
cgccttctgc cgacattaac agaagcacaa aaaatgtggt tgagtggtta tttaacagca 120
gctcaatcta cgtctgccga aggaacgcca gccgtttcta cagcagcgcc tgctcaaacg 180
aagcagacag tttcaaaaga tgtaacgatt ctttacggat cacaaacagg aaatgctcaa 240
ggacttgctg aaaatacagg caaaacgctt gaagcaaaag gttttaatgt aactgtatct 300
tctatgaatg atttcaaacc aaataattta aagaaacttg aaaatttatt aattgttgta 360
agtacacatg gagaaggaga gccgcctgat aacgccctgt ctttccacga atttcttcac 420
ggccgtcgag cgccaaaact tgagaatttc cgtttttctg tcttgtcgct tggagacagc 480
tcatacgaat ttttctgtca aaccggaaaa gaatttgatg tgcgcttagc agaacttggc 540
ggtgaaagac tgtatccgcg cgttgactgt gatttagatt ttgaagaacc cgcaaataaa 600
tggcttaaag gtgttattga cggattaagc gaagcgaaag gacacagcgc ttcggcagct 660
gttccggcgg aagctcctgc aggaacttca ccatattcaa gaacaaatcc ttttaaagca 720
gaagtgcttg agaacctaaa cttaaacggc cgcggatcaa ataaagaaac gcgacactta 780
gaactatctc tagaaggttc aggcttgacg tatgaaccag gagacagttt aggtatttat 840
cctgaaaatg acccggagct tgttgatctt cttcttaacg aattcaagtg ggatgcaagt 900
gaaagtgtaa cggttaataa agaaggagaa acgcgtcctc ttagagaagc gctaatctct 960
aattttgaaa ttaccgtttt aacaaaaccg cttttaaagc aagcagctga gcttactgga 1020
aacgataagt taaaagcgct tgtagaaaat cgcgaggaat taaaagcata cacacaaggc 1080
cgtgatgtga ttgacttagt tcgtgacttc ggcccatgga acgtatcggc acaagagttt 1140
gtagccattt tacgtaaaat gccagcgcgc ctttactcga ttgcaagcag tttatcagca 1200
aaccctgatg aagttcatct aacaatcgga gcggtacgct acgaagcgca tggacgcgag 1260
cgtaaaggtg tttgttcagt cctatgttca gaacgtttgc agccaggcga tacgattcct 1320
gtataccttc aaagcaataa aaactttaag cttcctcaag atcaagaaac gccaattatt 1380
atggtaggac ctggtacagg tgtggctccg ttccgctcat ttatgcaaga gcgtgaagaa 1440
acaggagcaa aaggcaagtc atggatgttc tttggagatc agcacttcgt aacagacttc 1500
ctttaccaaa cagaatggca aaagtggtta aaagatggcg tactgacaaa aatggacgtg 1560
gcgttttcac gcgatacaga agaaaaagta tacgtacaaa accgtatgct cgaacatagc 1620
aaagaattat tccagtggtt agaagaaggc gcatcttttt atgtgtgcgg agataaaaca 1680
aatatggcac gcgacgtgca caacacgcta gttgaaatta tcgaaacaga aggcaagatg 1740
agccgtgaac aggcggaagg ttaccttgct gaaatgaaga aacaaaaacg ttatcagcgt 1800
gatgtatact ga 1812
<210> 33
<211> 1278
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33299
<400> 33
ttgccggtta tacagggccc ctcgtcgtat aagcttaccg gtcacctgca aaagtttaga 60
gagaacccac tgggtttttt ggaaaacttg actcagtatg gagaaattgc aacatttaga 120
gttgcgcaca aacgctttta tgtaacgaga gaccctcaac ttattaaaga tgtagtcatt 180
acaaacagca aggcgtttca aaaaattaaa ctaacgcata tgtttaaaac gctgctaggt 240
gaagaaatgt tatggacaga tgaagcatta tacatgagcc ccattcaacc ctcacaactt 300
aaacaacatc taacttataa taaggaagca atcgcaaaaa taattgaaaa gcatatggaa 360
acgtgggaag aagggcagct tcgaacgata gtaaaagata ttagacaaat agtcgtagct 420
gtactccttc aacttgtatt tggcatttcg attgaggata aagataaaat tcactatgtt 480
caagcgctta tgagaaaaaa agaaaagtta ggcaaaattt atattcgtct acctttgcac 540
cagcctgact cagatgagca gctagaacaa ctcctttttg aacgcgttca aatgcgtgtt 600
caaaacaaaa ccgaagaaaa tgatttactg cagtatgtat tacattctca cggggaagac 660
atcgatgaaa gagaaatata tgaacagcta aattctatat tcctttcgat gtatgaaatg 720
attacgcacg tatgcagctg gtctattcat ttactgtctc aaaatactag ggagcacctt 780
cagctgcata aagaaattca agcctatgct agcggtgaat cactttcaac caaaaatctg 840
acctatatgc gcaaaataat tgctgaaagc atgaggctgt atccgcctct ttggctattt 900
gggcgtcaag cacgtgaaga tatacaaata gatggttaca gcattaaaaa aggggagatt 960
atgttaatta gcccttatat gatgcatcgc catgaagatt attttttaga gccaagtgaa 1020
tttttacctg accgttttga aaaaggaggg tccattgatg tcccaagcta tatgtatatg 1080
ccgcttggga ttgagcatca agcggaaaga ggcatggact atatcactga aatagtaact 1140
atttttctgt cagaaatgac aaagcgcttt ttatttcaac tgactaaggc cgagtctatt 1200
gctccgatgg cgggcgttat gctgaatatg aaagaagaac tgaatgtgaa tgttcacaaa 1260
gttcacacgc agtcttga 1278
<210> 34
<211> 1215
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33299
<400> 34
atgaaaaccg aaagagaaaa cggaatcgtc cgtcaagtga atacgattca aacaaaagaa 60
gagcgcttta atccttttac atggtatgaa gagatgagaa acagcgcgcc tgtgcaatgg 120
gatgaagaaa ggcaggtatg ggatgttttt cactatgacg gggtcaaaga agtgctggaa 180
caaaagaata ttttttcttc tgatcgaaga cctccacaaa accaaagaca aactgctcta 240
ggaacgagcc taattaatat tgatccgcct aagcatgctg aaatgagagc gcttgttaat 300
aaagctttta cgcctaaagc aatgaaagca tgggagccta aaattgcgcg tattaccgct 360
gaattattac aagaagttga gcatcttgaa gatattgata tagtcgagca tctttcctat 420
ccgcttccgg ttatggtaat tgccgatatt ttaggcgtgc cgatcgaaga tcagcgtcag 480
tttaaagatt ggtcggatat tatcgtagcg ggtccatcga ataatgaacg tgaaacgctc 540
gaaaaattgc agcaagagaa aatgaaagca aacgatgagt tagaaactta cttttatcga 600
atcattgaag aaaaacggac gcagccagga gatgatatta tttccgtgct tcttcaagca 660
aaagaagaag ggaagcagct aacggatgaa gaaatcgtcg ggttttccat tttgctgctg 720
attgcaggaa acgaaacaac tacgaactta atttcaaata caatttattg tttaatggaa 780
gataaggctt cttttgaaca cctcagacgt gagaaagagc ttttgccttc tgcgatcgaa 840
gaagttcttc gctatcgttc acccgttcag gctcttcacc gaatcgtaaa agaagatgtg 900
actcttgccg gtcagaaact aaaagcgggc gaacacgtcg ttccatggat gggatctgcg 960
catcgagatg ctcagtactt tgaagacccg gatgtattta aaatcgatcg aaagccaaat 1020
gtgcacatgg catttggaag aggcattcat ttttgcttag gagcaccgct tgctcgaata 1080
gaagcaaaaa ttatgctggc tgagctgatt gaccgctatc cgcagatgga ctggagccca 1140
gcatttgagt tgaagccgat tgaaagcacg tttgtgtacg ggttaaaaga attattaatt 1200
cgtaaacatg tataa 1215
<210> 35
<211> 1212
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33299
<400> 35
atgaacccaa aagcagtgaa aagagaaaat cgttatgcta acttaattcc tatgcaagaa 60
attaaatccg ttgaacagca gctttaccca tttgatattt ataacagttt gcgtcaagaa 120
gcacctattc gttacgatga aagccgaaac tgctgggatg tatttgatta tgaaacagta 180
aagtatattt taaaaaaccc gtcgcttttt tcatccaaac gagcaatgaa agaacgccaa 240
gaaagtatct taatgatgga tcctccaaaa catacaaaat tacgaaactt agtgaataaa 300
gcttttacac caagagctat tcagcacttg gagggtcata ttgaagagat tgcagattat 360
ttattagatg aagtaagcag caaaaaaaag tttgatatag tagaggattt tgctggacca 420
ctgccgatta ttgtaatagc agagctactg ggcgtaccca tacaagaccg agcactgttt 480
aaaaagtact ccgatgactt agtgagcggt tctgaaaata attcagacga agcctttgcg 540
aaaatgatgc aaaagcgtaa tgacggtgtg ctttttttac aagggtattt taaagaaatt 600
atcgctaagc gtcagaaaga taaacaagaa gatcttattt cgctgctttt agaagcagaa 660
attgacggcg agcatttaac agaagaggaa gtactggggt tttgtattct attgctagta 720
gcaggcaatg aaacaacaac aaacttaatt acgaacggag tccggtatat gacagaagat 780
gtagacgtgc agaatgaagt tcgtcaagac atatcgctag ttccgaatct cgtggaagaa 840
acgcttcgtt attatcctcc tattcaagcg attggacgca tagccgctga agatgtagaa 900
cttggagagt gtaaaattaa aaaagggcag caggtaatta gctgggcagc aagtgcaaat 960
agagattcag ctaagtttga acggtctaac acttttgtcg tacatcgtaa agcgaatcct 1020
cacgtaagct ttggatttgg cattcatttt tgcttaggag caccgctagc aagaatggaa 1080
gggaaaattg cctttgctaa attactagaa aaaggggaat tttccaaagt tcagaatcag 1140
tctttaaagc caatagacag cccttttgtt tttggtgtaa aaaaatacga aattactttt 1200
aataacgtct ga 1212
<210> 36
<211> 1215
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33299
<400> 36
atggctgtca aaaaagtgat taacttagct ccaataacgc ccatgaaaga attaaataca 60
aaagagaagt attttaatcc gtttccaatc tatgaaatgt ttcgagaaga aaatccggta 120
cgctatgatg aggaaagaaa gtgctgggat atctttttat atgaagatgt agactacgtt 180
cttaaacatc acgagctctt ttcttctcag cagcagcgaa tggtagaagg ggaaaactta 240
ttaagaatgg atcctccccg tcataaacaa atgcgcgatc tcgttaataa agcgtttacg 300
cctagagaaa ttaataatct tgctcctcgt attgaagaaa ttactaatca cttgctgcaa 360
aatgtcatag acaaaggaga aatgaaaatg attcatgact ttgccatgcc gcttccggtc 420
atcgtcatag ctgatttgtt aggcgttccg gccgaggaca gggaaaagtt taaggagtgg 480
tctgatatcc ttgtaaaagg agtaagtgaa aataagcctg aagcgtacga agcagtgaag 540
caggaacagc agattgcccg cgatgagtta aaggaatatc ttcatagaat tgtgcatata 600
agacgtttcg atcctaaaca agatatcata tcagctttac tttcatcaga agttgacgga 660
ggtaagcttg cagacagcga aatattagag ttcagtatgc ttctcttggc tgctggtaat 720
gaaacgacga caaacttaat tacaaatgga gtccgtctgc ttactgaaat accgagtctg 780
caaaaacaag tgaaaaatga tctttcactt gtgccaacta tggtggagga ggttctccgg 840
ttttatccac ctgtacaggc gccttcacgt attgccataa aagacgttga acttagaggt 900
aagacaataa gaaaagggga tgcggtgaca gcctgggtag catcagctaa ccgagatgaa 960
gctaaatttc ctcatgctaa tgtatttcaa gcggatcgta agcccaatca gcatcttact 1020
tttgggaaag gaattcattt ctgcttaggt gctcccttag cccgtctaga agcaaaaatt 1080
gcgtttgaag aaatgctaag taaatgtacg aatcttcaat taaaagaagg ttcattaata 1140
gaacgaactc cggcaccgca aatgtacggt gtggtagaat atcctgttac attttcagtg 1200
agcccttcat tatag 1215
<210> 37
<211> 1233
<212> DNA
<213> 巨大芽孢杆菌菌株DSM 33299
<400> 37
atgaacaaag aagtcattcc cgtgacagaa attccaaaat ttcaatcacg tgcagaagag 60
tttttcccta tccaatggta taaagaaatg ttaaacaatt cccccgttta ttttcatgaa 120
gaaacaaaca catggaatgt attccaatac gaacatgtta aacaagtctt gagcaactac 180
gaattttttt caagcgatgg acaaagaacc actatttttg ttggagataa tagcaagaaa 240
aaaagcacat ctccaatcac caaccttaca aacttagatc ctcctgacca ccggaaagcg 300
cgatctttgc ttgctgccgc ctttactcct cgcagtttaa agaactggga gccgcgaatc 360
aaacaaattg cagcagacct tgtagaagag acacaaaaga attcaactat taatattgtt 420
gaagatttgt cttctccttt tcctagttta gttatagcag atttattcgg cgtaccggta 480
aaagaccgat atcagttcaa aaagtgggta gatattcttt ttcagcctta tgaccaggaa 540
agattaaaag agattgaaca agaaaagcaa cgtgcaggag ctgaatattt tcaatatctc 600
tacccgattg tcgttgaaaa gcgctccaac ctttctgatg atattatttc agacttaatt 660
caagcggaag ttgatgggga aacattcaca gatgaagaaa ttgtgcacgc taccatgctg 720
cttttaggtg caggagttga aaccacaagt cacgccattg caaatatgtt ttattctttc 780
ttatatgacg acaagtcgtt atacagcgag ctgagaagca atagagaatt agctccaaaa 840
gcagtagaag aaatgcttcg atatcggttt catatctcca gacgagatcg cacggttaaa 900
caagataacg agctattagg cgttaagcta aaaaaaggag atgtcgtgat tgcgtggatg 960
agtgcatgta atatggatga aaatatgttt gaaaaccctt tttctgtaga cattcatcgt 1020
ccaactaata aaaagcactt aacattcgga aacggacctc atttttgttt gggcgcccct 1080
cttgcccgac ttgaaatgac aattatctta gaagcatttt tagaggcctt ttctcatatc 1140
gaaccatttg aaggttttga attagaaccg catttaacac cttcagctac gggtcaatct 1200
ttaacatact tgcccatgac ggtgtacaaa taa 1233

Claims (17)

1.一种制剂，其包含
-至少一种属于巨大芽孢杆菌(Baccillus megaterim)属的益生菌菌株，和
-多不饱和脂肪酸组分，其包含选自二十碳五烯酸(EPA)、二十二碳六烯酸(DHA)、花生四烯酸(ARA)、α亚麻酸、亚麻油酸、二十碳四烯酸、二十二碳五烯酸、亚油酸、γ-亚麻酸的至少一种ω-3或ω-6脂肪酸，
其中所述多不饱和脂肪酸组分包含ω-3或ω-6脂肪酸盐。
2.根据权利要求1的制剂，其中所述益生菌菌株以休眠形式或作为营养细胞存在。
3.根据权利要求1的制剂，其中所述ω-3或ω-6脂肪酸是游离脂肪酸、盐、天然甘油三酯、鱼油、磷脂酯或乙酯的形式。
4.根据前述权利要求中任一项的制剂，其中所述脂肪酸选自ω-3脂肪酸EPA和DHA，或其中ω-6脂肪酸组分为ARA。
5.根据前述权利要求中任一项的制剂，其还包含5-氨基酮戊酸。
6.根据前述权利要求中任一项的制剂，其中所述益生菌菌株选自以下的一种或多种：巨大芽孢杆菌DSM 32963，巨大芽孢杆菌DSM 33296或巨大芽孢杆菌DSM 33299。
7.根据前述权利要求中任一项的制剂，其中所述多不饱和脂肪酸组分包含ω-3脂肪酸盐，优选氨基酸盐，更优选选择自选自赖氨酸、精氨酸、鸟氨酸、胆碱及其混合物的碱性氨基酸，最优选赖氨酸。
8.根据前述权利要求中任一项的制剂，其中所述多不饱和脂肪酸组分包括包含至少一种磷脂和至少一种ω-3或ω-6脂肪酸的分散体的制剂。
9.根据前述权利要求中任一项的制剂，其中所述制剂包括用于延迟释放或肠或结肠释放的包衣。
10.根据前述权利要求中任一项的制剂作为饲料或食物补充剂的用途。
11.根据权利要求1至9中任一项的制剂在制备药物产品中的用途。
12.一种饲料或食品组合物，其包含根据权利要求1至9中任一项的制剂和至少一种其他饲料或食物成分，优选地选自蛋白质、碳水化合物、脂肪、其他益生菌、益生元、酶、维生素、免疫调节剂、代乳品、矿物质、氨基酸、抗球虫药、基于酸的产品、药物及其组合。
13.根据权利要求12的组合物，其用于改善动物或人类的肠道健康、心血管健康、心脏代谢健康、肺健康、关节健康、眼睛健康、精神健康、口腔健康或免疫健康的健康状态。
14.根据权利要求13的组合物，其用于通过以下一种或多种改善动物或人类的健康状态：
-经由其通过胃肠道微生物的产生增加宿主中以下脂质介质的总量：17-HDHA、14-HDHA、13-HDHA、7-HDHA、4-HDHA、18-HEPE、15-HEPE、12-HEPE、11-HEPE、5-HEPE、15-HETE、12-HETE、11-HETE、8-HETE、5-HETE、9-HODE、13-HODE、PDX、PD1、AT-PD1、MaR1、MaR2、LTB4、t-LTB4、RvD1-5、AT-RvD1、RvE1、RvE3、LXA4、LXA5、LXB4、LXB5，
-增加宿主中EPA的总量，
-增加宿主中DHA的总量。
15.根据权利要求1至9中任一项的制剂用于使用合适的基质或载体在皮肤、眼睛和口腔上的局部施用的用途。
16.根据权利要求15的用途，其中将所述制剂装载在包含纳米纤维素的预合成多相生物材料上和/或中，其中所述纳米纤维素是选自以下的细菌合成的纳米纤维素(BNC)：
-包含纤维素纤维或纳米晶须的网络的BNC，
-包含两个或更多个不同层的纤维素原纤维的BNC，其中每个层由来自不同微生物或来自在不同条件下培养的微生物的BNC组成，
-包含至少两个不同的纤维素网络的BNC或
-BNC复合材料，其进一步包含聚合物。
17.表现出以下特征的休眠形式的或作为营养细胞的巨大芽孢杆菌菌株用于SPM的生物技术生产的用途：
a)与根据SEQ ID NO：1或SEQ ID NO：2、SEQ ID NO：13或SEQ ID NO：14、SEQ ID NO：25或SEQ ID NO：26的多核苷酸序列具有至少99.5％、尤其是100％的序列同一性的16S rDNA序列；和/或
b)与根据SEQ ID NO：3、SEQ ID NO：15或SEQ ID NO：27的多核苷酸序列具有至少99.5％、尤其是100％的序列同一性的yqfD序列；和/或
c)与根据SEQ ID NO：4、SEQ ID NO：16、SEQ ID NO：28的多核苷酸序列具有至少99.5％、尤其是100％的序列同一性的gyrB序列；和/或
d)与根据SEQ ID NO：5、SEQ ID NO：17、SEQ ID NO：29的多核苷酸序列具有至少99.5％、尤其是100％的序列同一性的rpoB序列；和/或
e)与根据SEQ ID NO：6、SEQ ID NO：18、SEQ ID NO：30的多核苷酸序列具有至少99.5％、尤其是100％的序列同一性的groEL序列。
CN201980078589.8A 2018-11-30 2019-11-28 包含巨大芽孢杆菌属益生菌菌株和多不饱和脂肪酸组分的制剂 Pending CN113395908A (zh)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08298982A (ja) * 1995-05-02 1996-11-19 Aasu Giken:Kk 複合微生物製剤
US6107334A (en) * 1998-02-23 2000-08-22 Wake Forest University Dietary control of arachidonic acid metabolism
US20020055538A1 (en) * 2000-02-16 2002-05-09 Serhan Charles N. Aspirin-triggered lipid mediators
WO2018045004A1 (en) * 2016-08-30 2018-03-08 Agrinos AS Defined microbial compositions

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020188024A1 (en) * 2000-08-23 2002-12-12 Chilton Floyd H. Fatty acid-containing emulsion with increased bioavailability
ES2781863T3 (es) 2014-12-23 2020-09-08 Evonik Operations Gmbh Proceso para aumentar la estabilidad de una composición que comprende ácidos grasos omega-3 poliinsaturados
MX2017009180A (es) * 2015-01-15 2018-03-28 JeNaCell GmbH Biomateriales de nanocelulosa sintetizada bacterialmente y de fases múltiples, y método para producirlos.
WO2017041094A1 (en) 2015-09-03 2017-03-09 Solutex Na Llc Compositions comprising omega-3 fatty acids, 17-hdha and 18- hepe and methods of using same
EP3248467A1 (de) 2016-05-25 2017-11-29 Evonik Technochemie GmbH Verfahren zur herstellung einer zusammensetzung enthaltend omega-3-fettsäure-l-lysin-salze

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08298982A (ja) * 1995-05-02 1996-11-19 Aasu Giken:Kk 複合微生物製剤
US6107334A (en) * 1998-02-23 2000-08-22 Wake Forest University Dietary control of arachidonic acid metabolism
US20020055538A1 (en) * 2000-02-16 2002-05-09 Serhan Charles N. Aspirin-triggered lipid mediators
WO2018045004A1 (en) * 2016-08-30 2018-03-08 Agrinos AS Defined microbial compositions

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHING T HOU: "Biote chnology for fats and oils: newoxygenated fatty acids", NEW BIOTECHNOLOGY, vol. 26, no. 1, pages 2 - 10 *
CHING T HOU: "Effect of environmental factors on the production of oxygenated unsaturated fatty acids from linoleic acids by Bacillus megaterium ALA2", APPLIED MICROBIOLOGY AND BTIOTECHNOLOGY, no. 69, pages 463 - 468 *
MAI MIURA，等: "The Effect of 5-Aminolevulinic Acid on Cytochrome P450-Mediated Prodrug Activation", PLOS ONE, vol. 10, no. 7, pages 1 - 9 *

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